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Antibody to the common "a" determinant of hepatitis B surface antigen (HBsAg) protects against infection with hepatitis B virus. A number of variant surface antigens with amino acid substitutions within the "a" determinant have been described in patients around the world. Both wild type and variant HBsAgs were expressed in the yeast Pichia pastoris and the antigens were semi-purified and quantitated. The effect on antigenicity of these changes was investigated in a quantitative fashion using four monoclonal antibodies known to bind to different epitopes within the common "a" determinant. The results suggest that amino acid substitution of T131I, K141E and G145R and insertion of 3 amino acids between residues 123 and 124 markedly affect the antigenic structure of HBsAg. These substitutions and insertions in the viral envelope may lead to evasion of the virus neutralizing antibody response and also to reduce efficiency of detection by immunoassays used for diagnosis and blood-bank screening., (Copyright 2000 Wiley-Liss, Inc.)

Although a multitude of hepatitis B markers are available to discriminate different states of hepatitis B virus (HBV) infection, there remains a need to detect the HBV DNA itself. The HBV DNA test is the only test that can quantify the number of complete virions. As shown in this review, the diagnostic value of testing for HBV DNA depends on quantification, and there are situations in which in vitro amplification is necessary and others in which testing for HBV DNA without preamplification is best. Indications for analysis of HBV DNA include the early recognition of chronic hepatitis B, discrimination of different HBsAg carrier states, recognition of the evolution of variants, indication and follow-up of anti-viral therapy with interferon, and assessment of blood products for infectivity. In each of these cases quantification of HBV DNA improves the diagnosis of HBV infection and will become an important tool in the routine diagnosis of hepatitis B virus infection in the future.

The polymerase chain reaction was evaluated as a diagnostic tool in 72 chronic hepatitis B virus carriers. Hepatitis B virus DNA was detectable in the serum of HBsAg-positive virus carriers using aliquots as small as 100 al. The detection limit for cloned hepatitis B virus DNA was 100 ag. Primer pairs for different regions of the HBV genome resulted in different sensitivity. Detection of the amplified hepatitis B virus DNA by Southern blotting and subsequent scintillation counting or densitometry allowed a semiquantitative assay. Using several primer pairs in parallel for optimal detection, all HBeAg-positive HBsAg carriers, 80% of HBe antibody-positive symptomatic HBsAg carriers and 57% of asymptomatic HBe antibody-positive HBsAg carriers were found to have hepatitis B virus DNA in the serum. During antiviral therapy hepatitis B virus DNA disappeared by the polymerase chain reaction assay in patients who became HBeAg negative, but polymerase chain reaction detected a relapse earlier than did the conventional dot blot. Pre-S antigens were assayed in serum and liver samples from most chronic carriers by enzyme-linked immunosorbent assay and/or immunoblot. Although most viremic carriers were strongly positive for pre-S1 and pre-S2 antigens, some hepatitis B virus DNA-positive HBsAg carriers did not have detectable pre-S antigens, and vice versa. Our data show that assay of hepatitis B virus DNA in the serum by polymerase chain reaction is by far more proficient than by dot blot and that it cannot be replaced by serological assays of HBeAg or pre-S antigen.

The mRNAs of transiently expressed cytokine genes contain AUUUA-rich sequences in the 3' untranslated regions. In order to examine whether the AU-specific endoribonuclease V (EC 3.1.27.8) described previously by us transinactivates those mRNA species, we introduced a 51-nucleotide ATTTA sequence from tumor necrosis factor into the 3' untranslated region of beta-globin gene. Transcripts of that construct, synthesized in vitro, were prone to endoribonuclease V digestion at those AU-rich sequences. Stimulation of human macrophages with lipopolysaccharide resulted in a shift of the association state of the enzyme from the nuclear matrix-associated to the free form. This shift was strongly prevented by the hepatitis B surface antigen (HBsAg) and more weakly by hepatitis B nucleocapsid antigen and hepatitis B antigen of the X region. HBsAg and, to a lesser extent, hepatitis B nucleocapsid antigen and hepatitis B antigen of the X region inhibited the release of alpha interferon, tumor necrosis factor alpha, and granulocyte-macrophage colony stimulating factor, while it had no effect on interleukin-1 production from stimulated macrophages. Using the human hepatoma cell line PLC/PRF/5, we provide further experimental evidence that endoribonuclease V acts in trans as a posttranscriptional inactivator for nuclear matrix-associated cytokine transcripts. These results suggest that those cytokine transcripts which contain reiterated (overlapping) AUUUA sequences are degraded by nuclear matrix-associated endoribonuclease V. This degradation was comparably high in cells incubated with HBsAg or cells which produced this antigen.

The sera of 16 individuals chronically infected with the hepatitis D virus were analyzed for hepatitis B virus (HBV) markers. The majority of these patients had a non-replicative form of viral type B hepatitis as indicated by negative tests for HBeAg and HBV-DNA. Pre-S-encoded proteins were detected in 13/16 sera. Sera that were negative for polymerized serum albumin did also not contain pre-S1-encoded proteins. The presence of pre-S-encoded proteins is probably predominantly associated with 22-nm HBsAg forms present in large amounts in sera of individuals with chronic type D hepatitis.

The small and the middle surface proteins of hepatitis virus form either the virion or the 22 nm particle both of which are secreted. The large surface protein by itself remains cell bound in artificially transfected cell culture unless it is accompanied by an excess of the smaller protens. Its behavior in vivo is not yet well studied. Using specific monoclonal antibodies for immunoblotting, we found an abundance of small surface protein in the serum of chronic virus carriers and moderate amounts in the liver irrespective of viremia. The large surface protein was present in the serum and the liver of viremic carriers. In nonviremic carriers, the large protein was absent from serum, but in the liver a shorter form of the large protein was readily detectable. These findings suggest a complex regulatory mechanism of the viral surface protein depending on the expression of other viral gene products.

A study was initiated to assess the significance of antibody to hepatitis Be antigen (anti-HBe) in HBsAg-negative individuals. Anti-HBe was demonstrated in the majority of sera positive for anti-HBs and anti-HBc. All sera positive for anti-HBs and negative for anti-HBc and most sera positive for anti-HBc but negative for anti-HBs were anti-HBe-negative. This implies that the antibody response to HBeAg is of shorter duration than that to HBsAg and HBcAg. Anti-HBe may help to discriminate between various states of hepatitis B virus infection found to be associated with anti-HBc-positive but HBsAg- and anti-HBs-negative sera. 2 individuals were anti-HBe-positive but HBsAg-, anti-HBs- and anti-HBc-negative, this finding is not understood.

Sera of 85 asymptomatic HBsAg carriers found among blood donors were tested for anti-HBc titers and were retested 4 years later. The results were correlated with the histological findings of first and final biopsy. None of 75 HBsAg carriers with normal or minimally changed liver tissue, 71 of them anti-HBe positive, developed chronic inflammatory liver disease. 4 HBsAg carriers eliminated HBsAg from the serum after a 1-to 3-year HBsAg-carrier state and 2 developed antibody against HBsAg in the sequel. In 65 of 75 cases we found unchanged anti-HBc titers. The geometrical mean titer (GMT) was 1:7,800 in the first and 1:7,000 in the final examination with a range of 1:400 and 1:25,600 in the group of HBsAg carriers with normal liver, and was 1:14,200 and 1:10,300, respectively, with a range of 1:800 and 1:51,200 in HBsAg carriers with minimal changes. In both groups the decrease of anti-HBc concentration within 4 years was not significant. The group of 10 HBsAg carriers with chronic hepatitis did not differ from healthy HBsAg carriers in respect to anti-HBc titers. Anti-HBc titers varied between 1:6,400 and 1:25,600, the GMT was 1:13,700 and 1:12,800, respectively. It is speculated that in healthy HBsAg carriers shedding of serologically undetectable quantities of complete and/or defective HBcAg from liver cell nuclei which contain HBcAg not detectable by immunofluorescence maintain the production of anti-HBc.

The present paper describes the demonstration of d, y, w, and r HBsAg determinants in one serum. It was shown that there are two populations of HBsAg particles: HBsAg/ad and HBsAg/ady. All complete Dane particles were of subtype HBsAg/ady. Further characterization of HBsAg/ady particles did not reveal morphologic differences when they were compared with HBsAg/ad and HBsAg/ay particles. An HBsAg/ady phenotype may be the result of a double infection with hepatitis B viruses or exchanges of DNA sequences that determine HBsAg/ay and HBsAg/ad to form a new genotype.

Hepatitis B markers were studied in seven patients with reactivated liver disease. Reactivation of chronic type B hepatitis, as indicated by the reappearance of hepatitis B e antigen (HBeAg) in the serum, was characterised by the appearance of hepatitis B virus-DNA (HBV-DNA) in the serum. The expression of pre-S 1 encoded protein remained unchanged in five of seven patients, and poly-HSA as a marker for pre-S 2 encoded protein remained detectable in six of seven patients before and after reactivation of chronic hepatitis. The level of serum HBV-DNA correlated well with the level of liver enzymes, which rose from normal to various levels after reactivation of the liver disease. The data suggest that inflammatory activity of the liver disease is not related to the expression of pre-S encoded protein but to viral replication. Possibly pre-C and C-gene encoded antigens, which are produced together with viral nucleic acid and expressed on the surface of HBV-infected liver cells, play the key role in liver damage believed to be mediated by cytotoxic T cells.

Immunosuppressive therapy was stopped in 12 individuals positive for hepatitis Be antigen (HBeAg) and hepatitis B surface antigen (HBsAg) and in 4 individuals positive for HBsAg but negative for HBeAg. Discontinuation of immunosuppressive therapy in HBeAg-positive patients was always associated with a bout of hepatitis and elimination of HBeAg in 8/12 patients. One patient died from liver failure and 2 patients experienced a decompensation of their liver disease indicating that this approach might be harmful if used therapeutically. A bout of hepatitis was not noted in any of the individuals negative for HBeAg when the immunosuppressive therapy was stopped, implying that this event is not potentially harmful to the patient.

A study was undertaken to elucidate the etiology of HBsAg-negative chronic hepatitis. Form 37 individuals with HBsAg-negative chronic hepatitis, 11 had liver membrane autoantibody (LMA) and were thus classified as autoimmune. 6 patients had anti-HBc, 1 of which was also positive for LMA. The majority of individuals with HBsAg-negative chronic hepatitis had antibodies to hepatitis A antigen (anti-HAV), in general at low titer. We conclude from our data that hepatitis A and hepatitis B virus infections are unlikely to play a significant role in inducing or maintaining HBs-Ag-negative chronic hepatitis. The etiological role of non-A non-B hepatitis agent(s) is difficult to estimate and must await the detection of appropriate markers for type non-A non-B hepatitis.

Eighty-eight asymptomatic carriers of hepatitis B surface antigen (HBsAg) were followed with biochemical, serologic, histologic, and immunohistologic studies over a period of four years. None of the 78 HBsAg carriers with normal or minimally changed liver tissue, antibody to hepatitis B e antigen (HBeAg) in serum, and no intranuclear hepatitis B core antigen (HBcAg) developed a chronic inflammatory liver disease. Four individuals lost circulatory HBsAg, and at least two individuals terminated their HBsAg carrier state. Seven asymptomatic HBsAg carriers with chronic hepatitis were characterized by HBeAg in serum and intranuclear HBcAg. However, three HBsAg carriers with chronic hepatitis and an absence of intrahepatocellular HBcAg were positive for antibody to HBeAg over the observation period. The mechanism that leads to chronic hepatitis in these patients remains to be determined.

During a period of 3 years we observed 5 patients with chronic active hepatitis B and 2 with hepatitis B virus-induced liver cirrhosis who cleared HBsAg from their sera after 2-14 years of HBsAg carriership. 4 of them developed anti-HBs. After HBsAg clearance there was no evidence of persisting inflammatory activity within their livers. 5 of the 7 patients had been treated for 1-39 months with prednisolone, sometimes in combination with azathioprine. This therapy, however, had been stopped more than 3 years before these patients terminated their HBsAg carriership. Our observations indicate that even after long-standing chronic active hepatitis B or hepatitis B virus-induced liver cirrhosis HBsAg may be eliminated in a considerable number of patients.

Pre-S1 and pre-S2 encoded antigens of hepatitis B virus were localized in liver tissue using monoclonal antibodies. They were found to be exclusively expressed in the cytoplasm of liver cells. Cell bound pre-S1 encoded protein was often detected in patients with chronic liver disease and viremia. Only a small number of the HBsAg positive cells also contained pre-S1 antigen. There was no correlation with nuclear HBcAg. Livers of non-viremic HBsAg carriers contained many HBsAg expressing liver cells, that were frequently also positive for pre-S2 encoded protein but contained no detectable pre-S1 encoded protein at all. It remains open whether cell bound pre-S2 containing proteins of middle size have a significance for pathogenesis, as they are present in individuals with chronic liver disease as well as in healthy HBsAG carriers, and may be associated with both increased and normal liver enzymes. Cell bound pre-S1 antigen with viremia may, however, be involved in the maintenance of viremia and liver disease.

Liver biopsy material of 22 in the serum HBsAg positive patients was tested with the fluorescent antibody technique for the localization of HBcAg and HBsAg in the liver tissue. Comparative studies were done with the following tissue preparation techniques: Cryostat technique, freeze drying, freeze substitution, cold ethanol paraffin embedding technique (SAINTE MARIE) and isolated liver cells. The investigations revealed the following results: 1. No HB-components could be detected with the cold ethanol paraffin embedding technique and freeze substitution. 2. Using the cryostat technique HBsAg could be demonstrated in 16/22 (cytoplasmatic localization) and HBcAg in 8/22 (nuclear localization). 3. With freeze drying HBsAg and HBcAg could be found in the same cases. The excellent tissue preparation allowed a correct localization of the HB-components to the cell structure. 4. In comparison to cryostat sections in isolated liver cells HBcAg could be demonstrated in 11/16 and HBcAg in 8/8 cases.

Serological and immunofluorescent studies were performed in 3 patients with the simultaneous presence of HBsAg and anti-HBs in the serum. Our investigations revealed the following results: HBsAg and anti-HBs in the serum were of different subtypes. HBsAg in the serum and in the liver had the same subtype. HBsAg in the liver did not react with the circulating anti-HBs in the patient's own serum. HBsAg-anti-HBs immune complexes could not be detected in the serum or in the liver tissue. These phenomena can most readily be explained by at least 2 contacts with hepatitis-B-viruses of different subtypes, one leading to immune tolerance and the other to an immune response. In a follow-up study there was no evidence for a short-term change in HBsAg subtype. Unexplained remains the cause why one contact with HBV did lead to immune tolerance and a second to an immune response.

Sera of asymptomatic hepatitis B surface antigen (HBsAg) carriers were analyzed for the presence of pre-S-encoded proteins. Four individuals with biopsy-proven chronic hepatitis uniformly expressed pre-S1- and pre-S2-encoded proteins. Individuals who had histologically normal or largely normal livers were heterogeneous with respect to expression of pre-S-encoded proteins. This heterogeneous expression of pre-S-encoded proteins occurred most likely due to difference in serum HBsAg concentration. Alternatively differences in pre-S gene expression need to be considered. Clinically the study indicates that expression of pre-S domains in serum is unrelated to viremia or chronic liver disease.

The role of pre-S encoded viral surface proteins in acute hepatitis B virus infection is still poorly understood. Binding sites for polymerized human serum albumin have been found to be encoded by the pre-s2 region of the hepatitis B virus genome. Recently, murine monoclonal antibodies against pre-s1 and pre-s2 encoded hepatitis B virus gene products were generated and used for their specific detection in serum. In sera from patients with acute hepatitis B, pre-s1 and pre-s2 antigen occurred in 16 of 20 and 15 of 20 patients, respectively. In the initial stage of the disease, pre-S gene products correlated with binding sites for polymerized human serum albumin, but not with hepatitis B surface antigen. Subsequently, pre-s1 and pre-s2 antigens were cleared from the serum of patients with acute hepatitis B before binding sites for polymerized human serum albumin and hepatitis B surface antigen. Possibly, the early clearance of pre-S markers can be of prognostic value in acute hepatitis B. The mechanisms of the early clearance of the pre-S antigens in acute hepatitis B remain to be elucidated. However, elimination by immunologic mechanisms appears likely.

The present study was initiated to assess the risk of infection of medical personnel and family contacts of hemodialysis patients. It confirms previous reports that hemodialysis patients and staff members of hemodialysis units are at high risk of acquiring HBV infection. Approximately 25% of the household contacts had serological signs of past or ongoing HBV infections. The risk of infection of the family contact was independent of the mode of dialysis but increased if the contact was a spouse, assisted in dialysis, or if the index hemodialysis patient was HBsAg positive. There is an urgent need for active immunization to prevent spread of HBV related to hemodialysis.

This paper describes a "solid-phase"-radioimmunoassay for the demonstration of HBeAg and anti-HBe. The investigations revealed the following results: 1. HBeAg is positive in all patients with acute type B-hepatitis during the acute phase of illness. During the normal course of the disease HBeAg turns to negative followed by an anti-HBe lasting for several months. 2. Cases with a persistent virus B-replication as HBsAg-positive CPH, CAH or patients on hemodialysis are positive for HBeAg in their serum. By means of the fluorescent antibody technique these patients have demonstrable HBcAg and HBeAg in their liver biopsies. 3. Healthy HBsAg carriers are anti-HBe-positive in their serum. In their liver biopsies there are no signs of an on-going virus B-replication (HBsAg and HBeAg negative). 4. The radioimmunological determination of HBeAg and anti-HBe enables us to differentiate between the groups with HBsAg positive acute or chronic hepatitis and the group of healthy HBsAg-carriers.

The immunogenicity and efficacy of different commercially available vaccines against hepatitis B virus (HBV) has been demonstrated in a number of controlled vaccination trials throughout the world. However, the significance of pre-S proteins in hepatitis B vaccines is still controversially discussed. Therefore, a radioimmunoassay (RIA) was developed to detect pre-S proteins in HBsAg-carriers as well as in different hepatitis B vaccines. This study confirmed the high amount of pre-S proteins as well as poly-albumin receptors in purified Dane particles and sera of patients with acute hepatitis B. The currently available plasma derived vaccine H-B-Vax and the recombinant hepatitis B vaccines Gen H-B-Vax and Engerix B yielded negative results in pre-S- and poly-albumin-receptor-RIAs. The plasma derived vaccine Hevac B Pasteur included pre-S encoded proteins. Future vaccination trials in man have to evaluate the role of pre-S antigens and antibodies in the pathogenesis and the protection of hepatitis B infection including recombinant pre-S vaccines which are under development.

A study was undertaken to establish the risk of family contacts of HBsAg carriers acquiring a hepatitis B virus (HBV) infection. About one-third of all household contacts of asymptomatic HBsAg carriers had signs of past or ongoing HBV infection. Family contacts of HBsAg carriers with high numbers of circulating Dane particles were shown to have a higher risk of developing HBV infection than family contacts of HBsAg carriers without serological evidence of HBV synthesis. The probability of acquiring HBV infection was not different between spouses, parents, children, and brothers and sisters, respectively of asymptomatic HBsAg carriers.

A total of 32 individuals with HBsAg-positive and anti-delta-negative chronic hepatitis were treated with recombinant alpha-A interferon in phase I and phase II studies. In 5/32 patients HBsAg could be eliminated and in 19/32 individuals HBeAg became negative including all those who also eliminated HBsAg. Side-effects were tolerable in most patients and were readily reversible upon discontinuation of interferon therapy. In conclusion, treatment of HBsAg-positive chronic hepatitis with interferon seems to be a promising therapeutic approach. Future studies will have to establish the optimal dose, duration of treatment and factors predicting a favourable outcome of the treatment.

This paper gives the results for HBeAg and anti-HBe titers in chronic HBsAg carriers and patients with type B hepatitis using a "solid-phase" radioimmunoassay. In tumor and hemodialysis patients the HBeAg titers are statistically significant higher compared to the group of HBsAg positive CAH or CPH. High anti-HBe titers are a characteristic finding in "healthy" HBsAg carriers. On the other hand, there is a subgroup of HBsAg positive CAH with anti-HBe; although there are signs of an ongoing virus B replication these cases of CAH proceed sometimes to cirrhosis.

Anti-delta as an indicator of chronic delta infection was found in 7/180 HBsAg carriers. This indicates a low incidence of delta infection in Germany. The following risk factors to acquire delta infection were established: Mediterranean origin (3 cases) and transfusion of blood products (4 hemophiliacs). All anti-delta positive individuals had a biopsy proven chronic active hepatitis and the serological pattern: HBsAg, anti-HBc and anti-HBe positive without indicators of Dane particle synthesis (negative tests for HBeAg and DNA polymerase activity).

The HBsAg carrier state may present as chronic active hepatitis which may proceed to cirrhosis of the liver and to primary liver cell carcinoma. The large scale production of interferons made these substances available for long-term treatment. A deficiency in interferon production in chronic type B hepatitis presented the rational to treat this disease with interferon alpha-A. In this phase II-trial 3/31 patients eliminated HBsAg and 14/31 HBeAg. This was followed by normalisation of liver function tests and probably an improved prognosis. Efficiency of treatment was dependent on the interferon dose, the level of viral replication, the level of liver enzymes before treatment and concurrent infections (e. g. HIV infection). Reactivation occurred in five patients suggesting that the treatment period was too short in some individuals. Future studies will potentially improve efficiency by the modification of the interferon schedule and a better understanding of the mode of action of interferon.

A study was undertaken to assess the state of hepatitis B virus (HBV) infection in hemodialysis patients. From 97 hemodialysis patients tested, 51 were found to have at least one hepatitis B virus specific marker. 18 were HBsAg carriers, 12 of these carriers have to be regarded as infectious as judged from the presence of HBeAg and/or HBV-specific DNA polymerase activity in the serum. Antinuclear antibodies (ANA) were found in the sera of approximately 20% of the hemodialysis patients with a high prevalence in cases which lacked HBV markers. We conclude from our study that HBsAg-positive hemodialysis patients should be dialyzed in a separate unit and preferably served by personnel which is anti-HBs-positive. The question whether patients in which anti-HBc represents the only HBV marker should be separated is still open and needs further work. The role of non-A/non-B infection is difficult to determine and further studies are needed to elucidate this question.