glycoprotein-e2--hepatitis-c-virus and Chronic-Disease

Hepatitis C virus (HCV) causes persistent infection in most patients. To clarify the mechanisms underlying establishment of this persistent infection, nucleotide sequences of the E1/E2 region were characterized in 5 patients with acute and chronic HCV infection. We used direct DNA sequencing methods to identify the major sequence of HCV in each patient. Each HCV genome displayed a high frequency of nucleotide sequence variation in the hypervariable region (HVR) of E2. However, patient-specific conserved nucleotide sequences were identified in the E1/E2 region during the course of infection and conserved the higher-order protein structure. In the acute phase HCV infection, amino acid substitution in HVR-1 as the monthly rate of amino acids substitution per site (%) between each point exceeded 10.2%. In the chronic phase HCV infection, a significantly lower rate of amino acid substitution was observed in patients. The host immune responses to HVR-1 of each HCV isolates from all clinical courses were characterized using synthetic peptides and ELISA. One chronic patient serum (genotype 1b) did not react at all to its own HVR-1 peptides, however another patient (genotype 2b) reacted to all clinical course. These results indicated that HVR-1 might not always exhibit neutralizing epitopes of HCV infection. The sequence variation in HVR-1 may instead indicate the existence of various clones in acute phase infection and the adaption of these clones is thought to have caused persistent and chronic infection in each patient.

Topics: Acute Disease; Adult; Aged; Amino Acid Sequence; Amino Acid Substitution; Base Sequence; Chronic Disease; Female; Genetic Variation; Genotype; Hepacivirus; Humans; Male; Middle Aged; Molecular Sequence Data; Sequence Analysis, DNA; Viral Envelope Proteins; Viral Proteins

Infection with hepatitis C virus (HCV) generally progresses to chronic disease, although a minority of patients appear to clear viremia spontaneously. In this investigation, serum samples were analyzed for virological parameters, serum alanine aminotransferase (ALT) levels, and neutralizing antibody response against pseudotyped vesicular stomatitis virus (VSV) generated using chimeric envelope glycoprotein 1 (E1) or 2 (E2) of HCV. Testing of sequential serum samples that were collected beginning at the onset of acute-phase disease demonstrated intermittent viremia, elevated ALT levels, and detectable neutralization activity against VSV in 9 of 10 patients. Serum neutralization activity did not exhibit a correlation with the genotype of the infecting HCV or with virus load. On the other hand, patients with chronic HCV infection consistently had detectable amounts of virus present but no significant variation in ALT levels, and serum samples from a majority (>90%) of patients failed to show detectable neutralization activity.

Topics: Acute Disease; Adolescent; Adult; Alanine Transaminase; Chronic Disease; Female; Hepatitis C; Hepatitis C Antibodies; Humans; Male; RNA, Viral; Vesicular stomatitis Indiana virus; Viral Envelope Proteins

The antibody response to the hypervariable region of the E2 protein (HVR1) of hepatitis C virus (HCV) was studied in 5 patients who were infected by a common virus strain during an outbreak in a hemodialysis unit. Two patients resolved the infection, while 3 developed chronic HCV infection. For studying the antibody response to HVR1 during the early phase of infection, a Western blot assay using recombinant phage displaying HVR1 was developed. The 2 patients with resolving infection had a more rapid antibody response to HVR1 than did the patients developing chronic infection. Anti-HVR1 antibodies were repeatedly absent in 1 of the chronically infected patients. Antibodies to recombinant E2 protein occurred later than the anti-HVR1 antibodies and did not correlate with resolution of the infection. Thus, the present results suggest that early appearance of antibodies to the HVR1 may predict clearance of HCV infection.

Topics: Amino Acid Sequence; Antibodies, Viral; Bacteriophages; Blotting, Western; Chronic Disease; Cloning, Molecular; Enzyme-Linked Immunosorbent Assay; Hepacivirus; Hepatitis C; Humans; Immunoglobulin G; Molecular Sequence Data; Recombinant Proteins; Renal Dialysis; Viral Envelope Proteins

The viral variability of 5 hepatitis C virus (HCV)-infected immunocompromised patients was analyzed and compared with that in isolates from immunocompetent subjects. The patients were followed longitudinally with regard to changes in hypervariable region 1 (HVR1) of HCV using a direct DNA sequencing approach. For the immunocompromised patients, viral nucleotide sequence variability was markedly lower than in immunocompetent HCV-positive patients. For 1 agammaglobulinemic patient and 1 AIDS patient, no variation in the major amino acid sequence of HCV HVR1 could be observed, while another agammaglobulinemic patient exhibited transient variations and amino acid substitutions despite the lack of functioning humoral immune response. The study supports the general hypothesis of humoral immune selection as the main force of sequence variation in the HVR1 region but suggests that other selection mechanisms may contribute to modulation of the composition of the viral population.

Topics: Amino Acid Sequence; Base Sequence; Chronic Disease; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Humans; Immunocompromised Host; Molecular Sequence Data; Peptide Fragments; Viral Envelope Proteins

Epitopes of hypervariable region 1 (HVR1) were mapped by enzyme-linked immunosorbent assay using follow-up sera of patients, all of whom were infected with the same isolate of hepatitis C virus (HCV). Our results suggest that (i) an early appearance (up to month 13 postinfection) of antibodies directed to the N terminus of HVR1 is associated with acute self-limiting infections of HCV and (ii) isolate-independent antibodies which are mainly directed to the C terminus of HVR1 seem to persist in chronically infected patients. The relevance of HVR1-specific antibodies for neutralization was evaluated by characterization of a rabbit serum.

Topics: Acute Disease; Amino Acid Sequence; Animals; Chronic Disease; Epitope Mapping; Hepatitis C; Hepatitis C Antibodies; Humans; Molecular Sequence Data; Peptide Fragments; Rabbits; Viral Envelope Proteins

Acute and chronic Hepatitis C virus infections were investigated retrospectively in chimpanzees that had been infected from a single source. Anti-E1 and anti-E2 were detected in two of three chimpanzees with a chronic infection, but were first detected 1 to 2 years after inoculation. Sequence evolution of the E1 region in three animals over a period of 9 to 11 years revealed a mutation rate of 1.02 to 2.23 x 10(-3) base substitutions per site per year. The acute phase viremia levels in acute infections which resolved appeared to be at least 10-fold higher than during the acute phase of chronic infections. During chronic infections, the viral load fell rapidly after the acute phase and remained at very low levels for several years. After 4 to 6 years, the viral load and liver enzymes increased again, suggesting reactivation of the infection. There was no clear temporal relationship between sequence evolution of the E1 region, changes in viral load, and the production of antibodies to the envelope proteins.

Topics: Acute Disease; Animals; Base Sequence; Chronic Disease; Cloning, Molecular; DNA Primers; Evolution, Molecular; Female; Hepacivirus; Hepatitis C; Male; Pan troglodytes; Phylogeny; Polymerase Chain Reaction; RNA, Viral; Viral Envelope Proteins; Viremia

The significance of circulating antibody to hepatitis C virus (HCV) envelope glycoprotein 2 (E2)/nonstructural protein 1 (NS1) glycoprotein was studied in 83 patients with chronic HCV infection diagnosed by polymerase chain reaction (PCR). E2/NS1 antibody was quantitatively examined by a passive hemagglutination test using recombinant E2/NS1 glycoprotein encompassing amino acids 388 to 664 of the HCV-H strain. The results were correlated with clinical and virological features such as genotypes and viremic levels assessed by a competitive reverse-transcription PCR assay. E2/NS1 antibody was found in 73 patients (88%), and its occurrence was related to viremic levels. E2/NS1 antibody titers were low in asymptomatic HCV carriers with low levels of viral replication; 9 of 17 such patients tested positive for E2/NS1 antibody (53%), compared with 64 of 66 chronic hepatitis C patients (97%) (P < .01). A significant direct relationship was observed between viremic levels and E2/NS1 antibody titers (r = .52, P < .01). Of the 13 patients with low viremic levels of < 10(6) copies/mL, only 5 tested positive for E2/NS1 antibody (38%), whereas 68 of the 70 patients with viremic levels of > or = 10(6) copies/mL had it (97%) (P < .01). As for the relation to HCV genotypes, no difference was seen in E2/NS1 antibody titers among genotypes examined (1b, 2a, and 2b). These findings suggest that the E2/NS1 antibody tested exhibits no neutralizing activity in chronic HCV infection but may serve as a serological indicator of active virus replication.

Topics: Adult; Aged; Base Sequence; Chronic Disease; Female; Genotype; Hemagglutination Tests; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Humans; Male; Middle Aged; Molecular Sequence Data; Polymerase Chain Reaction; RNA, Viral; Viral Envelope Proteins; Viremia

To determine the clinical significance of viral quasispecies heterogeneity, 59 patients with chronic hepatitis C were studied using singlestranded conformational polymorphism (SSCP) analysis of the HCV E2 hypervariable region 1 (HVR1); of these, 48 were subsequently treated with interferon-alpha. The SSCP method was validated using clones of known nucleotide sequence. HVR1 was amplified in 54 of 59 (92%) patients. The median number of SSCP bands per sample was 6 (range: 2-12). Increased quasispecies heterogeneity correlated with the estimated duration of HCV infection (P < 0.05), parenteral-acquired HCV infection (vs. sporadic, P < 0.05), serum HCV RNA levels (P < 0.05), and HCV genotype 1 infection (P < 0.05), but not with age, serum AST, ALT, or Knodell score. Patients who had complete and sustained response to interferon-alpha (n = 11) had lower pre-treatment quasispecies heterogeneity compared to patients who had complete response with relapse (n = 18, P < 0.05) or no complete response (n = 16, P < 0.01). However, multivariate analysis revealed that HCV viremia was a stronger predictor of response to interferon-alpha. These findings indicate that the estimated duration of HCV carriage, serum HCV RNA levels, and HCV type 1 are important determinants for the evolution of HCV quasispecies heterogeneity; and that increased HCV quasispecies heterogeneity is another marker associated with a poor subsequent response to interferon-alpha.

Topics: Adult; Aged; Chronic Disease; Female; Genetic Heterogeneity; Hepacivirus; Hepatitis C; Humans; Male; Middle Aged; Polymerase Chain Reaction; Polymorphism, Single-Stranded Conformational; Reproducibility of Results; RNA, Viral; Viral Envelope Proteins

Antibody responses to the hepatitis C virus (HCV) envelope proteins E1 and E2 were analyzed using two original assays in sera from 86 patients in different stages of disease. A Western blot assay and an immunofluorescence assay (IFA) were developed using envelope proteins produced, respectively, in Escherichia coli and in CV1 cells infected with a recombinant SV40. As a third method, the INNO-LIA HCV Ab III assay including E2 synthetic peptides was used. Of 38 chronically infected patients positive for anti-E2 antibodies by IFA, 26 were positive in the Western blot assay (68%) and 25 in the INNO-LIA test (66%). Thus, the detection of anti-envelope antibodies is highly dependent on the antigen formulation, and a native glycosylated form of the proteins is probably needed for their efficient detection. This study shows that the antibody response to HCV envelope proteins depends on the phase of infection. A few acutely infected patients displayed a response to E1 or E2 (36% by Western blot, 7% by IFA), and these antibodies seem to develop in patients evolving toward chronicity. The high prevalence in chronically infected subjects (62% to E2 by Western blot, 90% by IFA), particularly in subjects with essential mixed cryoglobulinemia (68% and 100%), confirms that the resolution of infection involves more than these antibodies. The antienvelope response in patients treated with interferon was investigated, but no significant relationship was found between antibody level prior to treatment and the evolution of hepatitis. The detection of anti-envelope antibodies, therefore, is not predictive of the response to antiviral therapy.

Topics: Acute Disease; Animals; Cell Line; Chlorocebus aethiops; Chronic Disease; Cryoglobulinemia; Gene Expression; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Humans; Interferon alpha-2; Interferon-alpha; Recombinant Fusion Proteins; Recombinant Proteins; Viral Envelope Proteins

A routine screening test used in the diagnosis of hepatitis C virus (HCV) infection is the anti-HCV antibody (anti-HCV) test containing core, NS3, NS4, and NS5 antigens of HCV. When HCV infection occurs in immunocompromised hosts, antibody formation against core, NS3, or NS4 antigens may be weak in the presence of HCV viremia and cannot be detected by routine anti-HCV tests. This study proposed that in immunocompromised hosts such as patients with chronic renal failure (whose capacity to form antibodies is diminished), antibody formation against the E2 region would be preserved, because the E2/NS1 region of HCV is strongly immunogenic. The aim of this study is to evaluate the significance of anti-E2 in the diagnosis of HCV infection among patients on maintenance hemodialysis who are anti-HCV-negative, using a conventional third-generation enzyme immunoassay (EIA) kit. The E2/NS1 gene of HCV encoding the amino acid sequence 388-664 was molecularly cloned into a vector containing an SV 40 promotor and was expressed in Chinese Hamster ovary cells. Using this E2 protein, the anti-E2 test was performed by EIA on 100 patients on maintenance hemodialysis, and on 50 patients with chronic hepatitis C who were anti-HCV-positive, to evaluate the antigenecity of the E2 protein. Of the 100 hemodialysis patients, 15 (15.0%) tested anti-HCV-positive using a third generation anti-HCV ELISA kit. Of the 85 patients who tested negative for anti-HCV, nine (10.6%) were anti-E2-positive and six (66.7%) of these anti-E2 positive patients showed HCV RNA viremia by HCV reverse transcription-polymerase chain reaction. Fourty-two (84.0%) of 50 patients with chronic hepatitis C were anti-E2-positive. As a control group, we tested for anti-E2 among 30 blood donors who were anti-HCV-negative, and also among 85 patients with hepatocellular carcinoma who were anti-HCV-negative, but in both groups, none (0%) was anti-E2-positive. In conclusion, these data suggest that the E2 protein of HCV should be included in a diagnostic anti-HCV kit for the detection of HCV infection in immunocompromised patients.

Topics: Animals; Blotting, Southern; Chronic Disease; Cricetinae; DNA Probes; Enzyme-Linked Immunosorbent Assay; Female; Follow-Up Studies; Hepacivirus; Hepatitis Antibodies; Hepatitis C; Humans; Immunoblotting; Immunocompromised Host; Polymerase Chain Reaction; Renal Dialysis; RNA, Viral; Serologic Tests; Viral Envelope Proteins

The high genetic variability of the 5' end of the envelope protein-coding region E2 (HVR1 E2) of Hepatitis C Virus (HCV) RNA has been suggested by many authors to play an important role in both virus persistence and outcome of liver disease. We studied the relations between HVR1 E2 variability and HCV genotypes, HCV-RNA levels and liver disease in 8 chronic HCV carriers (5 males and 3 females, median age 41 years, followed-up for a mean period of 3 years). Four were healthy HCV carriers with persistently normal ALT levels and normal liver histology and 4 patients with chronic liver disease. In each patient, the HVR1 E2 variability of 2 serum HCV-RNA isolates obtained at least 12 months apart were evaluated by direct sequencing. Nucleotide and amino acid homologies ranged between 97.6%-57.1% and 92.8%-25% in healthy carriers and 95.2%-55.9% and 89.3%-32.1% in patients, respectively. We did not observe any correlation between HVR1 E2 heterogeneity and HCV genotypes, viraemia levels, presence and extent of liver necroinflammation. Our findings suggest that HVR1 E2 heterogeneity has no direct implications in hepatitis, pathogenesis but it could play a major role in virus persistence.

Topics: Adult; Base Sequence; Chronic Disease; DNA Primers; Enzyme-Linked Immunosorbent Assay; Female; Genotype; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Humans; Male; Middle Aged; Molecular Sequence Data; Polymerase Chain Reaction; Retrospective Studies; RNA, Viral; Sequence Homology, Amino Acid; Sequence Homology, Nucleic Acid; Viral Envelope Proteins; Viremia

The physical properties of hepatitis C virus (HCV) particles were determined by ultracentrifugation on 20-60% isopycnic sucrose density gradients. We report that (i) two populations of HCV particles were found in the sera of patients with chronic HCV infection [at high density (1.186-1.213 g/ml) and at low density (1.099-1.127 g/ml)], (ii) virus particles with high density values were associated with immunoglobulin, and (iii) virus particles with low density values accumulated base changes within a hypervariable region (HVR) of the E2 envelope domain of the RNA genome. The results indicate that base changes within the HVR of E2 lead to the accumulation of immunoglobulin-free virus particles. Therefore, these findings imply that persistent HCV infection is established as a consequence of sequence variation in the E2 envelope domain.

Topics: Amino Acid Sequence; Base Sequence; Chronic Disease; DNA, Viral; Hepacivirus; Hepatitis C; Humans; Immunoglobulins; Molecular Sequence Data; RNA, Viral; Ultracentrifugation; Viral Envelope Proteins; Virus Latency