Vaccine- and hepatitis B immune globulin-induced escape mutations of hepatitis B virus surface antigen.

Hepatitis B virus surface antigen (HBsAg) vaccination has been shown to be effective in preventing hepatitis B virus (HBV) infection. The protection is based on the induction of anti-HBs antibodies against a major cluster of antigenic epitopes of HBsAg, defined as the 'a' determinant region of small HBsAg. Prophylaxis of recurrent HBV infection in patients who have undergone liver transplantation for hepatitis B-related end-stage liver disease is achieved by the administration of hepatitis B immune globulins (HBIg) derived from HBsAg-vaccinated subjects. The anti-HBs-mediated immune pressure on HBV, however, seems to go along with the emergence and/or selection of immune escape HBV mutants that enable viral persistence in spite of adequate antibody titers. These HBsAg escape mutants harbor single or double point mutations that may significantly alter the immunological characteristics of HBsAg. Most escape mutations that influence HBsAg recognition by anti-HBs antibodies are located in the second 'a' determinant loop. Notably, HBsAg with an arginine replacement for glycine at amino acid 145 is considered the quintessential immune escape mutant because it has been isolated consistently in clinical samples of HBIg-treated individuals and vaccinated infants of chronically infected mothers. Direct binding studies with monoclonal antibodies demonstrated a more dramatic impact of this mutation on anti-HBs antibody recognition, compared with other point mutations in this antigenic domain. The clinical and epidemiological significance of these emerging HBsAg mutants will be a matter of research for years to come, especially as data available so far document that these mutants are viable and infectious strains. Strategies for vaccination programs and posttransplantation prophylaxis of recurrent hepatitis need to be developed that may prevent immune escape mutant HBV from spreading and to prevent these strains from becoming dominant during the next decennia.

Characterization of the reactivity pattern of murine monoclonal antibodies against wild-type hepatitis B surface antigen to G145R and other naturally occurring "a" loop escape mutations.

The hepatitis B surface antigen (HBsAg) "a" domain harbors major B-cell epitopes. Viruses with mutations in this region emerge after vaccination or during hepatitis B immune globulin (HBIg) prophylaxis. A strain with G145R replacement has been almost invariably isolated as a major escape mutant. We investigated mutant antigen-antibody interactions with direct binding assays. G145R and 16 other naturally occurring recombinant HBsAg mutants were expressed in mammalian Cos-1 cells. The reactivity of a panel of 28 murine anti-hepatitis B surface antigen (anti-HBs) monoclonal antibodies to mutant antigens was measured with enzyme immunoassay and expressed as percentage compared with the wild-type (wt) HBsAg signal for each antibody. All point-mutated proteins displayed diffuse intracellular immunofluorescent labeling corresponding to a secretory pathway. Monoclonal antibodies (mAbs) were classified according to different binding patterns. The effect of mutations on antibody binding differs depending on the amino acid involved and on the location within the "a" loop. As expected, most antibodies had absent or negligible binding (<40%), notably with residue 145 replacements. However, we identified antibodies that reacted with conformational epitopes but nevertheless had adequate reactivity (>40%) with all mutant antigens, including G145R. The effect of G145R was more pronounced than that of G145A. A subgroup of antibodies had substantially increased recognition (>120%) of antigens with mutations in the first loop. We demonstrated that antibodies can be selected or combined that react with all mutants investigated, including G145R. These data offer perspectives for improving anti-HBs-based protection against hepatitis B.

Localization of a unique hepatitis B virus epitope sheds new light on the structure of hepatitis B virus surface antigen.

In a search for monoclonal antibodies (MAbs) that can bind hepatitis B virus surface antigen (HBsAg) with amino acid substitutions in the immune dominant 'a' region (escape mutants) we investigated the epitope recognition site of the human MAb 4-7B. Pepscan analysis and experiments with alanine substitution as well as substitutions known from nature pointed to residues 178-186 in the small S protein with the amino acid sequence PFVQWFVGL (key amino acids in bold) as the minimal epitope. Single amino acid substitutions at positions 122(R/K)(d/y), 134(Y/F), 145(G/R), 148(T/A) and 160(K/R)(w/r), representing 'a' region variants in recombinant HBsAg COS-I cells, did not influence binding of MAb 4-7B. Synthetic peptides (residues 175-189) including the 4-7B epitope sequence were able to evoke an anti-HBs response in rabbits. According to established polypeptide models, the 4-7B epitope region is located in the lipid layer of 20 nm HBsAg particles. The present results, however, suggest that residues 178-186 are exposed on the surface of the 20 nm particle. This may change our view of the structure of HBsAg.

[The management of acute hepatitis C and needlestick accidents with hepatitis-C virus positive blood]. / Beleid bij acute hepatitis C en bij prikaccidenten met hepatitis-C-viruspositief bloed.

Acute hepatitis C is rarely diagnosed, in part because of its usually subclinical course. Infection with the hepatitis C virus (HCV) has a high chronicity rate, 70-90%. The risk of infection after a needlestick accident with HCV positive blood is 3-10%. There are no efficacious preventive measures regarding HCV infection but treatment with the antiviral drug interferon alpha during the acute phase of the disease has shown to significantly reduce the risk of subsequent chronic infection. It is advised to evaluate individuals who were exposed to infected blood by a needlestick accident regarding HCV transmission, and to offer interferon treatment to them in case they become HCV positive, as demonstrated with a positive serum HCV-RNA test.

[Hereditary haemochromatosis: recent developments in diagnostics]. / Hereditaire hemochromatose: recente ontwikkelingen in de diagnostiek.

Hereditary haemochromatosis (HHC), a condition of abnormal iron absorption, is among the commonest diseases in humans. Early diagnosis is vital, because the disease is easily treated by phlebotomy to remove iron. Recently, mutations were identified in a HLA-class I-like gene, designated HFE and possibly associated with HHC. It was found that 83% of unselected HHC patients and more than 90% of patients with a clear family history of HHC, were homozygous for a single point mutation resulting in an amino acid substitution (Cysteine282Tyrosine) in the HFE protein. Although these data look compelling, they are not definitive proof that HFE is the haemochromatosis gene, but identification of these mutations will be of value in early diagnosis of haemochromatosis. However, the questions when to use the assay for detection of the Cys282Tyr mutation and how to interpret the results are not fully clear yet. Therefore, caution is still advocated when using molecular testing.

[Optimal antiviral therapy of chronic hepatitis caused by hepatitis C virus]. / A hepatitis C vírus által elöidézett krónikus hepatitisek optimális antivirális kezelése.

Chronic HCV disease is a major health problem world-wide. The majority of HCV positive patients will develop progressive liver disease with a high risk for cirrhosis and hepatocellular carcinoma after 20 and 30 years respectively. Sustained response after a 6, month course of interferon therapy is about 30%. New insights in viral biology and virus-host interactions led to new guidelines for diagnosis and therapy. Seventy percent of anti-HCV positive persons with persistently normal ALAT levels are HCV-RNA positive, and 60-70% of these have chronic hepatitis histologically. Biochemical parameters alone are inadequate for diagnosis of the disease and evaluation of therapy. HCV-RNA positive patients with normal serum ALAT and chronic hepatitis histologically should be considered for antiviral therapy within the setting of clinical trials. Response rates to interferon have been doubled with 1 year treatment. Additional improvement may be achieved by higher dosage, especially in patients with hepatitis caused by genotype 1b viruses. Including viral and host parameters in therapeutic strategy may lead to selective adaptation of dosage in order to optimize interferon therapy and to further improve its cost-effectiveness.

Plasma glutathione S-transferase alpha 1-1 levels in patients with chronic liver disorders.

Recent data suggest that plasma levels of the phase II detoxification enzyme glutathione S-transferase alpha may be a sensitive indicator of hepatocellular integrity in acute liver disorders but little information is available in chronic hepatic disorders. Using a newly developed enzyme linked immunosorbent assay, glutathione S-transferase A1-1 (GSTA1-1) levels were measured in 279 plasma samples from patients with chronic liver disorders. Results were categorized as normal or elevated plasma GSTA1-1 and normal or elevated plasma aspartate aminotransferase (AST) levels. In 24 patients with alcoholic liver cirrhosis, plasma GSTA1-1 levels were not significantly different from a group of 350 healthy controls and only one patient (4%) had an elevated GSTA1-1 level while 10 (42%) patients had elevated AST activities. In samples from patients with primary biliary cirrhosis (n = 150), primary sclerosing cholangitis (n = 26) or chronic hepatitis (n = 79) significantly (P < 0.0001) elevated plasma GSTA1-1 concentrations were detected in 25 (17%), 7 (27%) and 17 (22%) of the samples, respectively. AST activities were increased in a higher percentage of samples in all three disorders: 89%, 88%, and 57%, respectively. Plasma GSTA1-1 and AST levels were significantly correlated (P < 0.005) in the above mentioned disorders but not in alcoholic liver cirrhosis. It is concluded that plasma GSTA1-1 is not a sensitive parameter for the detection of hepatocellular damage in chronic liver disorders.

Chronic hepatitis C virus disease: an evaluation of procedures for diagnosis and treatment.

Chronic hepatitis C virus (HCV) disease is a major health problem worldwide. HCV infection has a chronicity rate of about 70%. A high percentage of these patients have slowly progressive liver disease ultimately leading to cirrhosis and hepatocellular carcinoma. Increased understanding of the viral biology and host-virus interaction has induced new diagnostic and therapeutic criteria. Up to 70% of anti-HCV-positive subjects with persistently normal serum ALT have chronic hepatitis histologically. A normal ALT does not exclude viral replication and after therapy ALT does not correlate with viral clearance. Therefore, biochemical parameters alone do not adequately inform about disease severity and effect of the antiviral therapy. Monotherapy with interferon 3 times 3 MU/week for 6 months has an immediate response rate of ca. 35% with early relapse in at least half of the patients. Several trials have been conducted to improve results. A 1-year duration of therapy leads to a sustained response rate of approximately 40%. Absence of HCV-RNA early after start of therapy could enable selection of the patients who will most probably benefit from treatment. Viral load, genotype and quasi-species variability are correlated with disease severity, prognosis and outcome of therapy; these viral parameters may be incorporated in studies of more individualized therapeutic strategies.

Hepatitis C virus: biological and clinical consequences of genetic heterogeneity.

Hepatitis C Virus infection accounts for the majority of post-transfusion and sporadic hepatitis. In Western Europe, anti-HCV is detected in 0.4-1.5% of healthy blood donors. There is a high frequency of progressive chronic hepatitis, ranging from 50 to 80%, which leads to cirrhosis in 20-50% of patients after 10-20 years. Viremic patients with minimal biochemical abnormalities may have chronic liver disease histologically. There is growing evidence that virological features of HCV are associated with different clinical manifestations and response to therapy. The RNA genome consists of a 5' and 3' Untranslated Region, a structural domain encoding the core and envelope proteins, and a non-structural domain. Different HCV isolates show a high sequence heterogeneity, which has led to the classification of currently six genotypes and several subtypes. There is a marked difference in the geographic distribution of HCV genotypes, with types 1, 2 and 3a being most frequently found in western countries. In The Netherlands, subtype 1b accounts for approximately 60% of all cases of chronic HCV. Serologic diagnosis based on recombinant C-100 antigens (first generation immunoassays) only reliably detected type 1, due to the heterogeneity of the NS4 region; inclusion of more conserved proteins c22 and c33 (second generation assays) has largely improved sensitivity of anti-HCV testing. Genotype 1b is associated with more severe liver disease and with lower response rates for antiviral therapy, compared with types 2 and 3. Quasispecies nature and escape mutants may enable viral persistence and the development of chronic liver disease. As cross-reactivity between genotypes is unlikely, prevention of HCV disease may be dependent on the development of multivalent vaccines.

Local gastric and serum amoxicillin concentrations after different oral application forms.

The high recolonization rate after monotherapy of Helicobacter pylori-positive gastritis may be due to insufficient local drug concentrations. To investigate the role of local diffusion, we measured levels of amoxicillin, a drug with good in vitro activity against H. pylori, in the mucosa and serum. One gram of amoxicillin was given to healthy volunteers as a tablet (n = 6) or as water-dissolved, fizzing "Tab" (n = 6). Gastroscopy with biopsies from the antrum, corpus, and fundus was performed at 30, 60, and 90 min. Concentrations in the mucosa were measured after homogenization with the agar diffusion method using Bacillus subtilis as the biological indicator. Serum samples, taken basally and every 15 min, were analyzed by high-pressure liquid chromatography. Drug levels in the fundus and corpus remained far below those in the antrum for both application forms. The highest concentrations were reached after 30 min, with bactericidal levels in the antrum in two of six subjects who took the tablet form and five of six subjects who used Tabs. At 60 and 90 min, almost all values were below the MBC for 90% of the strains tested. The concentrations in serum, however, rose continuously, to reach a maximum after 75 or 90 min. These results show that incomplete elimination may be due to subbactericidal concentrations of antibiotics with high in vitro efficiency at the desired site of action in vivo and that local diffusion in the mucosa is essential for therapeutic effectiveness against H. pylori.