Utility of enzyme-linked immunosorbent assays to test core antigen in the diagnosis and antiviral therapy management of hepatitis C virus infections.

In this study, we evaluate the performance of the enzyme-linked immunosorbent assays (ELISAs) for HCV Ag detection in the diagnosis and antiviral therapy management of HCV infections. For the diagnosis of an active HCV infection, the limit of detection of HCV Ag corresponding to HCV RNA level was approximately 7300 IU/mL; the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of HCV-Ag were 88.96, 100, 100, and 91.33%, respectively. The Pearson's correlation coefficient between HCV Ag and HCV RNA was 0.891. All patients with negative HCV Ag at interferon-α2α/ribavirin therapy week 1 achieved a sustained viral response (SVR), and the PPV was 100%; whereas in patients with positive HCV Ag at therapy weeks 12, the NPV for achieving non-response (NR) was 100%. The results showed that ELISAs for HCV Ag detection could be cost effectively applied to diagnose and evaluate the response to antiviral therapy for HCV infections.

Frequent detection of HCV RNA and HCVcoreAg in stool of patients with chronic hepatitis C.

BACKGROUND AND OBJECTIVE: HCV is transmitted mainly by parenteral routes. However, unprotected anal intercourse has also been identified as a risk factor for HCV infection. HCV RNA can be detected in blood, saliva, and bile, but the presence of HCV in stool has not been investigated yet. STUDY DESIGN: Therefore, stool samples of 98 patients were collected prospectively. Specific HCV primers were used to identify samples positive for HCV RNA. HCV RNA-positive samples were tested for HCVcoreAg with the Architect HCVAg assay (Abbott). Presence of occult blood was investigated by the hemoCARE guajak test. Viral stability and infectivity of recombinant HCV particles was investigated in vitro by incubation of genotype 2a chimeric virus Jc1 with bile and stool suspensions. RESULTS: HCV RNA could be detected in 68 out of 98 stool samples from patients with chronic hepatitis C and 16 samples also tested positive for HCVcoreAg. Presence of HCV RNA in stool was more frequent in male than in female and in patients with low platelet counts but was not associated with the detection of occult blood. Stool suspensions and to a lesser extent bile reduced the in vitro infectivity of genotype 2a chimeric Jc1 virus even though infection of Huh7 cells was not completely abrogated. CONCLUSIONS: In summary, this study shows for the first time that HCV can frequently be detected in stool samples of chronically infected patients irrespective of occult bleeding. We suggest that stool can be a potential source for HCV infection and thus unprotected anal intercourse should be avoided.

Mass spectrometric detection of the amino acid sequence polymorphism of the hepatitis C virus antigen.

A method for detection and identification of the hepatitis C virus antigen (HCVcoreAg) in human serum with consideration for possible amino acid substitutions is proposed. The method is based on a combination of biospecific capturing and concentrating of the target protein on the surface of the chip for atomic force microscope (AFM chip) with subsequent protein identification by tandem mass spectrometric (MS/MS) analysis. Biospecific AFM-capturing of viral particles containing HCVcoreAg from serum samples was performed by use of AFM chips with monoclonal antibodies (anti-HCVcore) covalently immobilized on the surface. Biospecific complexes were registered and counted by AFM. Further MS/MS analysis allowed to reliably identify the HCVcoreAg in the complexes formed on the AFM chip surface. Analysis of MS/MS spectra, with the account taken of the possible polymorphisms in the amino acid sequence of the HCVcoreAg, enabled us to increase the number of identified peptides.

[Features of proliferative response of T-cells on hepatitis c virus antigens in healthy individuals contacting with this virus].

AIM: Evaluation of CD3+ and CD3-/CD56+ proliferative response on hepatitis C virus antigens in healthy medical workers. MATERIALS AND METHODS: The study included 15 medical workers with length of service of 2 and more years without common risk factors (blood and blood products transfusion, abdominal operations, invasive procedures, use of intravenous drugs). Control group consisted of 9 healthy individuals without risk factors. Peripheral mononuclears were isolated from blood and then incubated 72 hours in the presence of mitogen/PHA, Core+NS4 1b genotype HCV, NS4 HCV2a+3a genotypes or medium. Proliferative activity was registered by the presence of cell division marker ki67 by using FACS. RESULTS: The initial immunogram of medical workers differed from the control group by a significantly lower quantity of CD3+ lymphocytes, in particular CD3+/CD8+ population. Incubation with PHAresulted in a decrease of quantity of CD3+/ ki67+, CD4+/ki67+ and CD3-/CD56+/ki67+ in the medical workers group. Cultivation with HCV antigens resulted in a significant decrease of Treg (CD3+/CD25high/FoxP3+) and activated T-lymphocytes population in the case of stimulation by Core and NS4 1b genotype antigens. Analysis of cell response on virus antigens based on proliferative activity index detected significant differences only for CD8+/ki67+. Stimulation by Core and NS4 1b genotype antigens resulted in an increase of quantity of these cells whereas in the case of NS4 2a+3a genotypes their decrease was observed. CONCLUSION: The described changes may reflect exhaustion of cell reactivity due to extra antigen load in the group of medical workers, while differentiated immunologic shifts on hepatitis C viruses of various genotypes are noted.

MultisHRP-DNA-coated CMWNTs as signal labels for an ultrasensitive hepatitis C virus core antigen electrochemical immunosensor.

An ultrasensitive and selective electrochemical immunosensor was developed for the detection of hepatitis C virus (HCV) core antigen. The immunosensor consists of graphitized mesoporous carbon-methylene blue (GMCs-MB) nanocomposite as an electrode modified material and a horseradish peroxidase-DNA-coated carboxyl multi-wall carbon nanotubes (CMWNTs) as a secondary antibody layer. After modification of the electrode with GMCs-MB nanocomposite, Au nanoparticles were electrodeposited on to the electrode to immobilize the captured antibodies. The bridging probe and secondary antibodies linked to the CMWNTs, and DNA concatamers were obtained by hybridization of the biotin-tagged signal and auxiliary probes. Finally, streptavidin-horseradish peroxidases (HRP) were labeled on the secondary antibody layer via biotin-streptavidin system. The reduction current of MB were generated in the presence of hydrogen peroxide and monitored by square wave voltammetry. Under optimum conditions, the amperometric signal increased linearly with the core antigen concentration (0.25pgmL(-1) to 300pgmL(-1)). The immunosensor exhibites the detection limit as low as 0.01pgmL(-1) and it has a high selectivity. The new protocol showed acceptable stability and reproducibility, as well as favorable recovery for HCV core antigen in human serum. The proposed immunosensor has great potential for clinical applications.

Kinetics of hepatitis C viral RNA and HCV-antigen during dialysis sessions: evidence for differential viral load reduction on dialysis.

Hepatitis C infection is a common problem in dialysis units. The prevalence ranges from 3% to more than 50%. Several reports have described a variable reduction of HCV-RNA during hemodialysis treatment sessions. But so far nothing is known about the HCV antigenemia or the kinetics of the reduction of HCV-RNA and HCV antigenemia during these sessions. HCV-RNA was monitored using the VERSANT HCV bDNA assay 3.0 (Bayer Healthcare Diagnostics, Leverkusen, Germany) or the HCV-Monitor TaqMan (Roche Diagnostics). HCV antigenemia was tested by using Ortho-trac-C assay (Ortho Clinical Diagnostics, Neckargemünd, Germany). Kinetics of HCV-RNA were available in 15 dialysis sessions measured by bDNA assay and in 5 dialysis sessions measured by rt-PCR. Quantitative HCV-antigenemia was available in fourteen dialysis sessions. Not only HCV-RNA but as expected also the HCV-antigenemia fell during the dialysis session. However, while the average reduction of HCV-antigen appears steady and linear, the level of HCV-RNA seems to be stable during the first 3 hr of dialysis, and decreases rapidly during the last 2 hr. The results seem to be independent of the HCV-RNA detection method. The different kinetics of HCV RNA and HCV antigen load suggest that there are different mechanisms responsible for the reduction of the HCV antigen and HCV-RNA, respectively. Reduction of viral load during dialysis session indicates a potential benefit of dialysis in case of HCV associated antiviral therapy.

[Comparative analysis of hepatitis C virus core protein in the plasma and serum samples from HCV-infected blood donors and patients with hepatitis C].

The aim of the study was to develop a sensitive and specific method for revealing the direct marker of hepatitis C virus (HCV)--core protein in the serum and to test it in the laboratory setting. Experiments were made on plasma and serum samples from asymptomatic HCV-seropositive blood donors (n=65), patients with acute (AHC) and chronic (CHC) hepatitis C (n=295), and HCV-seronegative blood donors (n=20). The processing protocol for serum included their concentration by means of polyethylene glycol and subsequent treatments of pellets to detect core protein in free virions, nonenveloped nucleocapsids, and immune complexes. This allowed an assay to be developed for the detection of core protein, by using a sandwich ELISA. Inclusion of a combination of three original monoclonal antibodies into the sandwich could reveal in the samples core proteins of at least 3 genotypes of HCV (1, 2, and 3) with a sensitivity of 20 pg/ml in the majority of HCV-infected subjects. The results of determination of core protein and HCV RNA correlated with a high degree of sensitivity. To detect HCV in the blood of patients with AHC, it was shown to be sufficient to find freely circulating virions whereas an analysis of immune complexes should be included in cases of CHC to achieve more sensitivity. The findings are a basis for developing a test system for the diagnosis of hepatitis C, including its early stages before seroconversion and for determining a viral load during interferon therapy. Introduction of the method into practice increases the reliability of the diagnosis of hepatitis C and virus-free safety of blood transfusions.

Value of HCV antigen-antibody combined HCV assay in hepatitis C diagnosis.

Reduction of the window period of hepatitis C virus (HCV) infection represents an important goal in the transfusional and diagnostic settings. Currently, the detection of HCV infection relies on the use of immunoassays to detect viral antibodies. A new enzyme immunoassay (Monolisa HCV Ag-Ab ULTRA) designed to simultaneously detect circulating HCV antigen and anti-HCV antibodies has been developed by Bio-Rad and registered by the European Authorities. Several evaluations have been conducted in Europe to determine whether this new assay can improve early detection of HCV infection. Sensitivity studies included 130 HCV RNA positive/anti-HCV negative samples, 21 well documented seroconversion panels and 430 anti-HCV genotyped samples from France and Italy. Specificity has also been assessed in 15,302 non-selected blood donations and hospital samples. Studies have shown that Monolisa HCV Ag-Ab ULTRA assay has been able to detect 40-90 % of HCV RNA positive/anti-HCV negative samples collected in the window period, improving early detection of HCV when antibodies may be undetectable. The mean delay in detecting HCV infection between HCV-RNA and this new test was found to be 5 days, reducing the window period by an average of 37 days. All samples collected after seroconversion were detected with the HCV Ag-Ab ULTRA assay. The specificity analyzed in 15,302 random blood donations and hospital samples was estimated at 99.86 %. Although less sensitive than NAT (71 % of HCV RNA positive/anti-HCV negative in window period), this assay could be a reasonable alternative when NAT cannot be used for reasons such as cost, organization, emergency or logistic difficulties.

Seroprevalencia de las hepatitis virales en población general representativa de una zona básica de salud urbana en Castilla y León / Seroprevalence of viral hepatitis in a representative general population of an urban public health area in Castilla y León (Spain)

Introducción. Las hepatitis virales constituyen uno de los principales problemas sociosanitarios y económicos a nivel mundial por lo que precisan un estrecho control epidemiológico. Métodos. En el presente trabajo estudiamos la seroprevalencia de las hepatitis virales una muestra representativa de la población de una zona básica de salud urbana en Valladolid (España). Resultados. La prevalencia de anticuerpos anti-VHA (AcVHA) fue del 52%, de HBcAc del 8,2%, de AcVHC del 1,1%, de AcVHE 0,8% y AcVHG 5,8%. La prevalencia de AcVHA, HBcAc y AcVHG aumenta significativamente con la edad (p < 0,005 en todos los casos). En menores de 20 años la prevalencia de AcVHA es del 3,8%, HBcAc < 0,28% y AcVHG 1,3%. En el grupo de edad de 20-39 años, la seroprevalencia frente al VHA se asocia con niveles educativos bajos (p 5 0,009) y con el nacimiento en otras provincias (p 5 0,016). La seroprevalencia de HBcAc se asocia principalmente con hospitalizaciones anteriores a 1990 (p 5 0,002; OR: 3,32 [1,48-7,42]), realización del servicio militar obligatorio anterior a 1990 (p < 0,0001; OR: 37,33 [3,68-378,03]) y prácticas de acupuntura (p 5 0,018; OR: 57,75 [26,17-127,42]). La seroprevalencia frente a VHG se asocia con hospitalizaciones antes de 1990 (p 5 0,019; OR: 2,969 [1,154-7,639]). Los seropositivos frente a VHC tenían antecedentes de transfusiones (2 casos) hospitalización (1 caso) o drogadicción (1 caso). De los seropositivos frente a VHE sólo un caso tenía antecedentes de viaje a zona endémica para VHE. Conclusiones. Nuestro estudio muestra que las seroprevalencias de las hepatitis virales en una muestra representativa de población urbana de Castilla y León son similares a las seroprevalencias obtenidas en el resto de España y de los países desarrollados, inferior a la observada en los estudios realizados en España en los últimos 20 años consecuencia de las medidas profilácticas adoptadas (AU)

Introduction. Viral hepatitis is a major social, health and economic problem worldwide, requiring strict epidemiological control. Methods. This study presents the viral hepatitis seroprevalence in a representative sample from an urban health care area in Valladolid (Spain). Results. Antibody prevalence was as follows: anti-HAV 52%; anti-HBc, 8.2%; anti-HCV, 1.1%; anti-HEV, 0.8%; and anti-HGV 5.8%. Prevalence of anti-HAV, anti-HBc and anti-HGV increased significantly with age (P < 0.005 in all cases). In individuals younger than 20, prevalence of anti-HAV was 3.8%, anti-HBc < 0.28% and anti-HGV 1.3%. In the 20-39 year-old group, seroprevalence against HAV was associated with low educational levels (P 5 0.009) and with birth in other provinces (P 5 0.016). Anti-HBc seroprevalence was mainly associated with three factors: prior hospitalization before 1990 (P 5 0.002; OR 3.32 [1.48-7.42]); compulsory military service before 1990 (P < 0.0001; OR 37.33 [3.68-378.03]); and acupuncture treatments (P 5 0.018; OR 57.75 [26.17-127.42]). Seroprevalence against HGV was associated with hospitalizations before 1990 (P 5 0.019; OR 5 2.969 [1.154-7.639]). Seropositive status to HCV revealed a transfusion history (2 cases), hospitalization (1 case) or drug addiction (1 case). Only one case among those seropositive to HEV had a history of a prior trip to a HEV-endemic area. Conclusions. Our study shows that the seroprevalences of viral hepatitis in a representative sample of urban population of Castille and Leon are similar to the seroprevalences observed in the rest of Spain and other developed countries, lower than the ones observed in the studies performed in Spain in the last 20 years due to the measures of prophylaxis that werw taken (AU)

Preparation of hepatitis C virus structural and non-structural protein fragments and studies of their immunogenicity.

Plasmids pQE-60 and pQE-30 containing 6 x His-tag sequence were used for expression of fragments of HCV structural and non-structural proteins in Escherichia coli (E. coli). The following fragments were used: core (1-98 aa), NS3 (202-482 aa), and tetramer of hypervariable region 1 (HVR1) of E2 protein. The constructed plasmids directed high levels of expression of HCV proteins in E. coli JM109. After purification by the metal-affinity chromatography on nickel-nitrilotriacetic acid (Ni-NTA) agarose, the His-tagged HCV proteins were used for immunization of BALB/c mice. All three proteins were able to induce high levels of specific antibodies and, in the case of the NS3 and HVR1 tetramer, also to mount vigorous cell-proliferating responses. High immunogenicity of the tested fragments of HCV proteins shows them as good candidates for inclusion into the future HCV vaccine preparations.

Proteomic profiling of lipid droplet proteins in hepatoma cell lines expressing hepatitis C virus core protein.

Hepatitis C virus (HCV) core protein has been suggested to play crucial roles in the pathogeneses of liver steatosis and hepatocellular carcinomas due to HCV infection. Intracellular HCV core protein is localized mainly in lipid droplets, in which the core protein should exert its significant biological/pathological functions. In this study, we performed comparative proteomic analysis of lipid droplet proteins in core-expressing and non-expressing hepatoma cell lines. We identified 38 proteins in the lipid droplet fraction of core-expressing (Hep39) cells and 30 proteins in that of non-expressing (Hepswx) cells by 1-D-SDS-PAGE/MALDI-TOF mass spectrometry (MS) or direct nanoflow liquid chromatography-MS/MS. Interestingly, the lipid droplet fraction of Hep39 cells had an apparently lower content of adipose differentiation-related protein and a much higher content of TIP47 than that of Hepswx cells, suggesting the participation of the core protein in lipid droplet biogenesis in HCV-infected cells. Another distinct feature is that proteins involved in RNA metabolism, particularly DEAD box protein 1 and DEAD box protein 3, were detected in the lipid droplet fraction of Hep39 cells. These results suggest that lipid droplets containing HCV core protein may participate in the RNA metabolism of the host and/or HCV, affecting the pathopoiesis and/or virus replication/production in HCV-infected cells.

[Late spontaneous elimination of HCV-RNA from mononuclear cells derived from peripheral blood in patients with chronic hepatitis C]. / Pózna samoistna eliminacja HCV-RNA z komórek jednojadrzastych krwi obwodowej po skutecznym leczeniu przewleklego zapalenia watroby typu C.

THE AIM: To follow persistence of HCV-RNA in PBMC in patients with chronic hepatitis C (CHC). To estimate the influence of this phenomenon on the cellular immune response of peripheral blood lymphocytes. MATERIAL AND METHODS: 8 HCV-RNA in PBMC positive children, with undetectable serum HCV-RNA after antiviral treatment, have been examined every 2-3 years. The amount of IFN-gamma, IL-12 and IL-18 secreted by PBMC obtained from the children after stimulation with phytohemagglutinin (PHA) was measured. RESULTS: Spontaneous elimination of HCV-RNA from PBMC in 2 to 6 years after treatment was found in all children. In two children HCV-RNA detectable both in serum and in PBMC, without recurrence of hepatitis, was found in single examination. PBMC containing HCV-RNA secreted more IFN-gamma than PBMC lacking it (1221 +/- 458 pg/ml vs. 651 +/- 147 pg/ml; p=0.009, similar correlation was revealed with the regard of IL-12: 21.8 +/- 12.3 pg/ml vs. 5,6 +/- 3,3 pg/ml respectively; p=0.009. Production and release of IL-18 were not correlated with HCV-RNA persistence (p=0.12). CONCLUSION: Patients with CHC and persistence of HCV-RNA in PBMC require longitudinal follow-up in the respect of possible reseroconversion. PBMC containing HCV-RNA reveal enhanced cellular immune response, which most probably effects in spontaneous elimination of the virus.

Ultra-sensitive class I tetramer analysis reveals previously undetectable populations of antiviral CD8+ T cells.

A major breakthrough in cellular immunology has been the development of HLA class I tetramers to analyze CD8(+) T cell responses. However, in many situations, including persistent virus infection, specific T cell responses are rarely detected using this technology. This raises the question of whether such responses are 'deleted' (or 'exhausted') or present below the conventional detection limit for class I tetramer staining. In particular, persistent hepatitis C virus (HCV) infection is characterized by very weak or apparently absent specific CD8(+) T cell responses, even though they are readily detectable in acute disease. Therefore, we assessed the use of anti-PE-labeled magnetic beads to enrich tetramer-positive HCV-specific T cells and identify previously undetectable populations. Using the enrichment technique, HCV-specific T cells could be detected in the majority of infected individuals, whereas these responses were not detected using conventional tetramer staining (8/15 vs. 1/15; p=0.01). Magnetic enrichment could reliably detect very rare HCV-specific responses at frequencies of >0.0011% of CD8(+) T cells (approximately 1/million PBMC), and phenotypic analysis of these rare populations was possible. Therefore, this direct ex vivo technique revealed the persistence of very low frequencies of virus-specific CD8(+) T cells during chronic virus infection and is readily transferable to the study of other viral, self- or tumor-specific T cells.

Purification of recombinant proteins with high isoelectric points.

The use of recombinant antigens is essential for the construction of robust and sensitive diagnostic assays. A critical step in the preparation of recombinant antigens is protein purification. Purification problems may be very different for related structural proteins expressed in the same host or for the same protein expressed in different hosts, because the biochemical characteristics of a recombinant protein, expressed in a heterologous system, are unique. In this chapter we make a brief introduction to protein purification procedures and we present a quick purification process suitable for the isolation of recombinant protein having high isoelectric points encoding non-conformational epitopes.

Genetic alterations in hepatocellular carcinomas: association between loss of chromosome 4q and p53 gene mutations.

The major risk factors for hepatocellular carcinomas (HCC) in high incidence areas include infection with hepatitis B and C viruses (HBV, HCV) and exposure to aflatoxin. Genetic alterations in 24 liver resection specimens from Shanghai and Qidong were studied. Hepatitis B virus was integrated in all patient samples, and a null phenotype for the GSTM1 enzyme was present in 63% of patients. Alteration of p53 was present in 95% (23/24) of cases: mutations of the p53 gene in 12 HCC, p53 overexpression in 13 and loss of heterozygosity (LOH) of chromosome 17p in 17. All seven HCCs with a p53 mutation from Qidong and three of five from Shanghai had the aflatoxin-associated point mutation with a G to T transversion at codon 249, position 3. No HCC had microsatellite instability. LOH of chromosome 4q, 1p, 16q and 13q was present in 50%, 46%, 42% and 38%, respectively, and 4q was preferentially lost in HCCs containing a p53 mutation: LOH of 4q was present in 75% (9/12) of HCC with, but only 25% (3/12) of HCC without, a p53 gene mutation (P = 0.01). These data indicate a possible interaction between p53 gene mutation and 4q loss in the pathogenesis of HCC.

In situ simultaneous detection of hepatitis C virus RNA and hepatitis C virus-related antigens in hepatocellular carcinoma.

BACKGROUND: The overwhelming evidence that chronic infection with the hepatitis C virus (HCV) is an important cause of hepatocellular carcinoma (HCC) is based on epidemiologic, case-control, and cohort studies as well as laboratory investigations. To address better the pathogenesis of HCV infection at the single-cell level, the authors developed a specific reproducible method for the simultaneous detection of HCV specific sequences and antigens in liver tissue, using a combination of nonradioactive in situ hybridization and immunohistochemistry. METHODS: After immunohistochemical staining of the liver sections for E2/NS-1, C22-3, C33c, C100-3, and NS-5 antigens with immunogold-silver technique, in situ hybridization was performed on the same sections using digoxigenin-labeled HCV 5' NonCoding specific probes. The hybridization signal was detected by an antidigoxigenin, Fab fragment-alkaline phosphatase conjugate. This simultaneous detection permitted the subcellular localization of HCV RNA and antigens with excellent preservation of tissue morphology and absence of background staining. In addition, the types and percentages of cells harboring HCV in tissue could be determined. RESULTS: The in situ detection of HCV showed positive signals in both cancerous and noncancerous areas of liver tissue in six of six HCV-infected patients with HCC and in none of four controls, including three HCV negative HCC patients and one patient with epithelioid hemangioendothelioma. Two classes of infected cells were distinguished throughout the liver: (1) cells containing large amounts of negative-stranded HCV RNA, which were probably undergoing active viral replication; and (2) cells displaying positive-stranded HCV RNA only, with unpredictable levels of viral replication. Both types expressed core, envelope, and NS-3, -4, and -5 proteins. HCV RNA and antigens were exclusively cytoplasmic. Detection of viral proteins was highly predictive of the presence of large amounts of HCV RNA in the same cell. Fewer HCV positive cells were consistently demonstrated in the cancerous area. CONCLUSIONS: These findings support the contention that HCV infects hepatocytes and replicates in them, even after their malignant transformation.

Excreción de porfirinas e infección crónica por virus C de la hepatitis / Porphyrin excretion and chronic infection by hepatitis C virus

Eighteen patients with cirrhosis Child-Pough A, eight infected with hepatitis C virus, were studied. Urinary excretion of Ù aminolevulinic acid, porphobilinogen, coproporphyrins, uroporphyrins and fecal excretion of coproporphyrins and protoporphyrins were measured. Red blood cell protoporphyrin was also measured. There were no differences in the measured parameters between patients with or without hepatitis C virus infection. No patient had uroporphyrin excretion values over the normal range. Some patients had slight elevations in some parameters, but always below the values observed in porphyrias. In these group of patients, hepatitis C virus infection of its associated liver diasease do not cause detectable alterations in porphyrin metabolism

Characterisation of a panel of monoclonal antibodies raised against recombinant HCV core protein.

Hepatitis C virus (HCV) has as yet no practical culture system so any antigen or antibody studies must be carried out using recombinant antigens. In this study, HCV core sequence was amplified by PCR, inserted into pRSET, and expressed in E. coli. The resultant core protein was purified using nickel affinity chromatography which bound the six histidine tag attached to the N-terminus of the protein. After elution in imidazole buffer, the core protein was used to immunise Balb/c mice and monoclonal antibodies against HCV core were raised. Six monoclonals were examined in a variety of assays. All of them recognised the p27 kDa protein which they were raised against and 2D2 and 3D7 recognised the core component of an HCV Recombinant Immunoblot Assay (RIBA). None of the antibodies recognised the linear peptides in an Innolia HCV assay. 2D2 showed cytoplasmic granular staining in 1-5% of cells in frozen section of HCV infected livers. Cross-competition assays between themselves and human anti-HCV core positive sera divided the antibodies into two main groups (I and II), with a sub-division of group I into a and b. Group I antibodies were unable to be inhibited by human anti-HCV sera whereas group II antibodies were inhibited by these sera (up to 62%). Epitopes recognised by all the monoclonals were probably conformational with the group I epitope being located within the first 105 amino acids of the core sequence and the group II epitope between amino acids 105 and 160.

Evaluation of hepatitis C virus protein epitopes for vaccine development.

Infection with hepatitis C virus (HCV) leads to viral persistence and chronic disease in a very high proportion of cases, despite a broad immunological response to viral proteins. These responses may thwarted by the high rate of mutation, which leads to the generation of 'escape' variants of HCV that persist as a quasi-species in infected individuals. The specificity of the immuno response of infected patients suggests that responses directed at certain viral epitopes may be associated with less aggressive disease and, possibly, good interferon response and virus clearance. The identification of such epitopes may hold the key for future development both of prophylactic and therapeutic vaccines.

Identification of hepatitis C virus by immunoelectron microscopy.

Sequencing of the hepatitis C virus (HCV) has provided a better understanding of the natural history, immunology, and epidemiology of this virus. However, the morphology of HCV has not been definitively characterized. In this study, through a sequence of concentration processes, virus-like particles were isolated from human serum and liver tissue, visualized by transmission electron microscopy and identified as hepatitis C virion by immunoelectron microscopy. Spherical flavi-like virus particles, approximately 70 nm in diameter, were observed in the fraction with 1.04-1.12 g ml-1 sucrose density and bound to immunogold particles with monoclonal antibodies (mAb) against hepatitis C. The nucleocapsid of the particles, which were 50 nm in diameter, appeared to be icosahedral in structure and surrounded by an envelope covered with surface projections. A 'tadpole' form of particles was also observed. The findings indicate that the low buoyant density in sucrose and the morphological features of the hepatitis C virion are consistent with the characteristics of flaviviruses and pestiviruses.