Lack of protective immunity against reinfection with hepatitis C virus.

Some individuals infected with hepatitis C virus (HCV) experience multiple episodes of acute hepatitis. It is unclear whether these episodes are due to reinfection with HCV or to reactivation of the original virus infection. Markers of viral replication and host immunity were studied in five chimpanzees sequentially inoculated over a period of 3 years with different HCV strains of proven infectivity. Each rechallenge of a convalescent chimpanzee with the same or a different HCV strain resulted in the reappearance of viremia, which was due to infection with the subsequent challenge virus. The evidence indicates that HCV infection does not elicit protective immunity against reinfection with homologous or heterologous strains, which raises concerns for the development of effective vaccines against HCV.

Localization of hepatitis C virus antigen(s) by immunohistochemistry on fixed-embedded liver tissue.

Using two sources of primary antibodies, we immunohistochemically stained hepatitis C virus-related antigen(s) on fixed-embedded liver specimens. These antigens were localized in the cytoplasm of hepatocytes. The results obtained serologically correlated well with immunohistochemistry.

IgM-antibody response to hepatitis C virus antigens in acute and chronic post-transfusion non-A, non-B hepatitis.

A specific IgM solid-phase enzyme-linked immunoassay for the diagnosis of a recent infection by hepatitis C virus (HCV) was developed. The assay utilizes a structural antigen encoded by sequences at the 5' end of HCV (core region) and non-structural (NS) antigens encoded by the NS-3 (33c) and NS-4 (c100-3) regions of the HCV genome. Serial serum samples from several clinically diagnosed post-transfusion non-A, non-B hepatitis patients were analyzed for anti-HCV IgM. This antibody was frequently but transiently detected. Anti-HCV core IgM was more frequently detected than anti-c100-3 or anti-33c IgM. In individuals who resolved their HCV infection or progressed to chronicity, anti-HCV IgM was produced transiently at or near the onset of clinically diagnosed acute hepatitis.

Expression of HCV envelope proteins and the serological utility of the anti-E2 immune response.

The 5' end of the hepatitis C virus (HCV) genome encodes structural proteins of the virion. The first gene encodes a highly basic core protein. Immediately downstream of the core gene are regions which encode the envelope proteins (E1 and E2) of the virus. Artificial expression and secretion of immunologically active envelope proteins have proven to be a substantial challenge due to the high degree of glycosylation and the existence of certain hydrophobic domains contained within these sequences. Bacterial cell expression of recombinant HCV envelope proteins results in products that are not glycosylated and are poorly immunogenic. Emphasis has shifted to the use of mammalian cell lines (human embryonic kidney [HEK] and Chinese hamster ovary [CHO] cells) for the expression of glycosylated, immunologically active envelope proteins. Using HEK cells, E1 is expressed intracellularly but is not secreted from the cells. When E1 is cloned in fusion with a C-terminal truncated E2 protein, both proteins are detected intracellularly; however, only E2 is secreted. When the E1/E2 processing site is interrupted by constructing deletion mutants, the unprocessed E1/E2 fusion protein can be secreted from the cells. Quantifiable expression and secretion of a truncated E2 protein is now possible using CHO cells and SV40-based vectors. The HCV E2 glycoprotein expressed from CHO cells is highly antigenic; a strong humoral response to this antigen develops in persons infected with HCV. Antibodies to E2 are found in 95% of patients with detectable HCV RNA in their sera. The presence of antibodies to E2 is not indicative of viral clearance and therefore the role these antibodies play in protective immunity, if any, is unclear.

Purine nucleoside and purine base concentrations in bovine thyroid and plasma.

Concentrations of eight purine nucleosides and bases in bovine thyroid and plasma were determined by high pressure liquid chromatography. Plasma purines were metabolized to uric acid in the absence of inhibitors. The concentrations of these purines were 10-100 times greater in thyroid tissue than in plasma.

Detection and distribution of hepatitis C-specific antigens in naturally infected liver.

Hepatitis C virus antigen expression was examined using peptide antibodies in liver tissue taken at biopsy from four chronic carriers of hepatitis C virus. Hepatitis C virus antigens E2/NS1, NS3, NS4 and NS5 were widespread in unfixed frozen liver sections and were present as distinct granules or foci within the cytoplasm of hepatocytes and in infiltrating lymphocytes in portal tracts. Fixation of frozen sections with 1% formalin improved the histological appearance of the tissue section without reducing the sensitivity of antigen detection. However, in tissue sections fixed in acetone, chloroform, carbon tetrachloride or methyl carnoys, detection of all hepatocyte-specific hepatitis C virus antigens was significantly reduced. Dual immunostaining of liver sections for lymphocyte cluster of differentiation markers and hepatitis C virus antigens determined that a high proportion of cluster of differentiation 20-positive B cells and cluster of differentiation 4-and cluster of differentiation 8-positive T cells, predominant in lymphoid aggregates, were positive for hepatitis C virus antigens.

Localization of hepatitis C virus (HCV) antigen by immunohistochemistry on fixed-embedded liver tissue.

An immunohistochemical method of staining HCV-related antigens in fixed-embedded liver biopsies is described. Two primary antisera were used: 1) a high titre anti-HCV human IgG separated from an anti-HCV positive serum; and 2) rabbit anti-HCV antibodies. In our experience this method proves to be reproducible and shows a good correlation with serologic results.

Antibodies to recombinant and synthetic peptides derived from the hepatitis C virus genome in long-term-studied patients with posttransfusion hepatitis C.

Eight of 13 Swedish patients (62%), studied prospectively, who developed posttransfusion non-A, non-B hepatitis (PT-NANBH) had earlier been found to seroconvert for antibodies to hepatitis C virus (anti-HCV) c100-3 in the first-generation anti-HCV enzyme-linked immunosorbent assay 1-18 (mean, 8) weeks after onset of hepatitis. By using a second-generation test utilizing antigens encoded by the core NS3 and NS4 region of HCV, a further four patients non-reactive to c100-3 (NS4) were found to seroconvert. Thus 12 of 13 (92%) Swedish patients with PT-NANBH were shown to have HCV infection. In addition, the serologic reactivity for several individual synthetic peptides and/or recombinant HCV proteins was studied in seven anti-HCV c100-3 seroconverts studied long-term after onset of acute PT-HCV infection. No special patterns were found that could differentiate patients who recovered from those who developed chronic HCV infection. It was concluded that the addition of new recombinant antigens derived from the core and NS3 region to c100-3 (NS4) both improved the sensitivity of the anti-HCV test and shortened the window phase to seroconversion.

Immunohistochemical detection of the NS4 antigen of hepatitis C virus and its relation to histopathology.

The immunohistochemical localization of the hepatitis C virus (HCV) nonstructural antigen 4 (NS4) was investigated in formalin-fixed human liver biopsy samples taken from 10 patients who were anti-HCV positive. NS4 was detected within the cytoplasm of hepatocytes in all HCV-positive patients studied, but not in the mononuclear cell infiltrates, bile duct epithelium, or endothelial cells. A high proportion of hepatocytes appeared positive, but the staining intensity was variable. After a coded histological evaluation of the liver tissue, the pattern of liver injury was shown to have no significant correlation with antigen-positive hepatocytes, and no direct relationship was observed between the distribution of antigen-positive hepatocytes and areas of hepatocyte necrosis. The staining pattern was considered to be specific because liver samples from patients chronically infected with hepatitis B virus or from uninfected individuals were negative. Furthermore, no staining was noted when either preimmune rabbit serum or anti-NS4 adsorbed against the specific synthetic peptide was substituted for the primary antibody.

Detection of the hepatitis C virus genome in acute and chronic experimental infection in chimpanzees.

In order to gain an understanding of the relationship of various markers of hepatitis C virus (HCV) infection in acute and chronic cases of the disease, serial blood samples obtained from chimpanzees before and after infection with HCV were analyzed for the presence of the HCV genome by using polymerase chain reaction (PCR) amplification of cDNA (cDNA PCR) synthesized from plasma- and serum-derived RNA. In a chimpanzee with acute hepatitis C, signals detectable by cDNA PCR appeared 1 week before characteristic ultrastructural changes visualized by electron microscopy, persisted throughout the peak alanine aminotransferase levels, and diminished with the disappearance of alterations visualized by electron microscopy. This was in contrast to the results obtained from chimpanzees with chronic HCV infection, in which the HCV genome was consistently detectable for up to 10 years after infection. The results indicate the usefulness of detection of HCV RNA by cDNA PCR as a sensitive and semiquantitative method for monitoring the course of HCV infection and as a potential marker for differentiating between chronic and acute cases of disease.

Risk of liver disease in HCV-seropositive kidney transplant recipients.

OBJECTIVE: This study determined whether renal allograft recipients with antibodies to hepatitis C virus (HCV) at the time of transplantation experienced increased morbidity or mortality from hepatitis, liver disease, or hepatocellular carcinoma compared with patients without anti-HCV. SUMMARY BACKGROUND DATA: Chronic liver disease is a cause of significant morbidity and mortality after kidney transplantation and the contribution of HCV to this problem has not been determined. The recent characterization of the HCV genome has resulted in the development of screening tests for antibody to HCV, allowing the identification of end-stage renal disease patients with anti-HCV who are candidates for transplantation. The risk to these patients for the development of hepatic complications after subsequent transplantation is unknown. METHODS: Archived sera obtained from 163 kidney transplant recipients at the time of transplantation were tested for anti-HCV using the Abbott HCV 2.0 second-generation test system. Sera containing anti-HCV were further analyzed for reactivity against specific HCV recombinant proteins, including core, NS3 (c33c), and NS4 (c100-3), to determine whether a pattern could be identified in patients with hepatic complications. The follow-up of all patients was current (mean length of follow-up was 33 months) to identify patients with hepatic complications. All patients had previously been tested for HBSAg. RESULTS: Twenty-nine patients (18%) had anti-HCV and three (1.8%) had HBSAg. Forty-five patients (28% of total) had transient elevations of AST or ALT without subsequent evidence of liver disease. Three patients had a syndrome of acute hepatitis. Chronic liver disease developed in only six patients (3.6%) after transplantation. Four had anti-HCV only, one had HBSAg only, and one was positive for both. However, of the 29 patients with anti-HCV, chronic liver disease developed in 5 (17%), including 1 patient who was positive for HBSAg. No patient had hepatocellular carcinoma. CONCLUSIONS: Perturbations of liver function were common in the kidney transplant recipients studied, most were self-limited, and few were associated with evidence of viral hepatitis. The risk of developing

Hypervariable 5'-terminus of hepatitis C virus E2/NS1 encodes antigenically distinct variants.

Synthetic peptides representing sequences encoded at the 5'-terminus of E2/NS1 in hepatitis C virus (HCV) were constructed. Peptides synthesized based on the sequences of four distinct HCV isolates were used to develop enzyme immunoassays (EIAs) for detection of antibodies in chronic HCV patients and in HCV-infected plasma donors. HCV sequence-specific antibodies were detected among patients with chronic HCV from the United States and Italy at frequencies of 22.2% and 55.8%, respectively. Similarly, sequence-specific antibodies were detected in 54.6% of U.S. and 55.6% of Japanese commercial plasma donors who had previous evidence of HCV exposure. Our data support earlier findings of geographic variability among HCV variants. The region encoded by amino acids (aa) 380-436 was shown to contain at least one variant-specific and one conserved epitope. The data further indicate that a majority of patients chronically infected with HCV (58.1% U.S., 68.8% Italy) have antibodies directed to the 5'-terminus of the E2/NS1 gene product. We conclude that genotypic variability within the E2/NS1 gene of HCV results in antigenically distinct variants.

Significance of anti-E2 in the diagnosis of HCV infection in patients on maintenance hemodialysis: anti-E2 is frequently detected among anti-HCV antibody-negative patients.

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.

Persistent hepatitis C virus infection after liver transplantation: clinical and virological features.

We report a prospective clinical and virological study of 18 patients undergoing orthotopic liver transplantation, selected because of hepatitis C virus (HCV) RNA positivity before transplantation. Nine of the 18 patients (50%) developed chronic active hepatitis (CAH) in liver allografts during the first year posttransplantation; hepatitis was first observed between 6 and 25 weeks posttransplantation. HCV viremia was measured for all patients before transplantation and on posttransplantation days 3, 7, and 14, and months 1, 6, 12, and 24 to 41, by quantitative competitive RNA polymerase chain reaction (QC-PCR). HCV RNA levels on posttransplantation days 3, 7, and 14 were significantly higher among patients who subsequently developed CAH versus those who did not (P < .02 by t-test and Mann-Whitney test on all three dates). However, HCV RNA levels in sera obtained at 1, 6, and 12 months posttransplantation did not correlate with CAH at 1 year or with HCV genotype determined in posttransplantation sera. At least two serial liver biopsy specimens from each patient were stained for HCV nonstructural 4 (NS4) antigen by immunohistochemistry. The intensity of cytoplasmic staining of NS4 antigen was significantly higher for specimens with CAH versus those without CAH (P = .028 by chi 2). Three patients developed bridging fibrosis in liver allografts during the first year after transplantation; all three patients had intense (3+) immunostaining for NS4 antigen, and the infecting genotypes were 1a, 1b, and 1a plus 1b, respectively. In summary, the 18 patients all developed high-titer viremia by 1 month after liver transplantation, whereas CAH developed in 50% of allografts during the first year after transplantation.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of antibodies to hepatitis C virus in U.S. blood donors.

An enzyme immunoassay (EIA) which utilizes a solid phase coated with a recombinant antigen (c100-3) derived from the hepatitis C virus (HCV) genome was evaluated for efficacy in the detection of antibodies to HCV (anti-HCV). The sensitivity of the antibody test was demonstrated by the detection of anti-HCV in a well-characterized panel of human specimens known to contain the infectious agent of non-A, non-B hepatitis. The specificity of the anti-HCV test was evaluated by testing 6,118 serum specimens from volunteer blood donors considered to be at low risk for exposure to HCV. The specificity of the anti-HCV EIA was demonstrated to be 99.56%, since 6,069 of 6,096 specimens from this low-risk group were nonreactive. A total of 49 (0.80%) of the 6,118 specimens were repeatedly reactive in the test, and 22 (46.81%) of the 47 specimens available for additional testing were confirmed as positive for antibodies to HCV c100-3. Among commercial plasma donors, 390 (10.49%) of 3,718 specimens were repeatedly reactive in the EIA. A total of 375 (97.40%) of the 385 specimens available for further testing were confirmed as positive. These limited data indicate that the prevalence of antibodies to HCV is 0.36% (22 confirmed positives among 6,118 specimens) among volunteer blood donors and 10.08% (375 confirmed positives among 3,718 specimens) among commercial plasma donors. The importance of confirmatory testing is discussed.