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Hepatitis B virus DNA was extracted from serial serum samples of a hepatitis B surface antigen-negative patient with antibodies to the core protein as the only marker of an infection with hepatitis B virus. This patient showed no symptoms of hepatic injury. Sequencing of the amplified viral DNA demonstrated multiple amino acid changes clustering in surface-exposed regions of the surface protein. Synthesis and association of the middle (M) and small (S) surface proteins could be shown in vitro. The variant surface antigens were recognized neither by monoclonal antibodies to the surface antigen nor by the vaccinee's sera. Consequences for hepatitis B surface antigen testing and vaccine development are discussed.

The prevalence and time course of the occurrence of antibodies to the hepatitis B virus polymerase (anti-HBpol) were investigated in acutely and in chronically HBV-infected individuals by using recombinant HBpol protein for Western blot analysis. One group consisted of 19 patients who were acutely infected and recovered completely. Five of these patients (26%, 69 serum samples examined) exhibited anti-HBpol. Among those anti-HBpol positive patients, recovery from the disease was combined with a complete loss of this antibody. In contrast, in a second group of 15 individuals who developed chronic hepatitis B, 13 (87%, 102 serum samples examined) had anti-HBpol during the acute phase of the disease. The difference between the anti-HBpol prevalence rates of the two patient groups is statistically significant (Exact Fisher test, P < .002), implying that the occurrence of anti-HBpol may be indicative of a potential chronic course of hepatitis B. Remarkably, anti-HBpol was found in one case of a clinically suspected hepatitis B in which no other serological HBV parameters were found. This serum sample was positive in HBV PCR, supporting a possible diagnostic value of anti-HBpol.

On the basis of published sequence data the preS1 attachment region of hepatitis B virus (HBV) appears to be highly variable. Using a novel method for rapid DNA sequencing by the polymerase chain reaction we screened 34 HBV DNA-positive sera for mutations in a variable part of the preS1 region of the HBV genome. The sequence data were used to analyse potential chains of infection, and strongly supported the expected routes of HBV transmission among patient groups. Furthermore, sequence comparisons permitted sub-genotyping of the viruses. In the 22 cases of subtype adw, we found a very low number of point mutations. This shows that the attachment site of HBV is more highly conserved than that of other blood-transmissible viruses such as human immunodeficiency virus or hepatitis C virus.

The diagnostical significance of the large hepatitis B surface protein with its preS1 attachment site and of anti-preS antibodies are not yet well known. We investigated the epitope of the preS1 attachment site to see whether it is a marker of viremia and whether antibodies against it occur in convalescents and vaccinees. For comparison, sera were also tested for the presence and relative amount of a preS2 epitope. The epitopes were detected by binding to specific monoclonal antibodies (mAb MA18/7 for the preS1 epitope and mAb Q19/10 for the preS2 epitope) at the solid phase of a sandwich enzyme-linked immunosorbent assay. Antibody against the preS1 epitope was detected by inhibition of binding to mAb MA18/7. This mAb inhibits attachment of preS1 antigen to hepatocytes and reacts with a subtype-independent sequential epitope at the surface of hepatitis B virus between amino acid 29-36. This preS1 epitope occurs in most hepatitis B surface antigen (HBsAg) carriers, irrespective of viremia. Free preS2 epitope Q19/10 is present in samples with more than 8 micrograms/ml total HBsAg and it is masked in sera with less HBsAg. Antibodies which compete with mAb MA18/7 for its viral preS1 epitope occur in one third of HBsAg carriers who were negative for hepatitis B e antigen. It also occurs in one third of convalescents and in most good responders to plasma-derived vaccines.

Using direct solid-phase radioimmunoassay (dSP-RIA) which was shown to be specific and about 10000 times more sensitive than immunodiffusion and even 10 times more sensitive than the radioimmunoprecipitation test used by us before, anti-HBs was detected in 90% of hepatitis B convalescents who were followed up for a one year observation period and who had eliminated HBs antigen from their sera (60/68). In 13% of these patients (8/60 anti-HBs was only temporarily detectable. In 50% of all patients who developed antibody within a two to twelve months observation period (n = 151), anti-HBs appeared within three to four months after height of disease. In 16.5% of patients with HBs antigen negative hepatitis (32/193) anti-HBs was observed to develop during convalescence indicating a type B infection despite a negative finding for HBs antigen in the acute stage of disease. Using dSP-RIA anti-HBs was found in 5.5% of voluntary blood donors (n = 1036), in 20.2% of hospitalized patients without clinical hepatitis (n = 252), and in 30.6% of hospital staff (n = 304). Anti-HBs increases with age: in 14 year olds we found 0.5%, in 60 year olds, 14%. The fact that at least 70% of the anti-HBs positive normal persons and medical staff groups had no history of hepatitis illustrates the frequency and importance of subclinical hepatitis B infections.

A method for quantitative standardization of the DNA hybridization assay for hepatitis B virus (HBV) DNA protein complex in serum is described. This method was used to determine the titer of HBV DNA in various groups of subjects with HB surface antigen (HBsAg) in order to ascertain its accuracy as an index of infectivity. The method's detection limit was 10(5) genome equivalents or 0.3 pg DNA per ml. Titers of 5 X 10(7) to 5 X 10(8) genome equivalents per ml were found to be typical for persistent massive viremia, which occurred more frequently in symptomatic (30 of 48) than in asymptomatic (24 of 72) carriers positive for HBe antigen (HBeAg). Moderate viremia (10(5)-5 X 10(7)) was usually found in patients eliminating the virus from the blood. Patients with resolving acute hepatitis B were frequently positive at the onset (18 of 26) with moderate titers, but became negative within several weeks. In 11 patients who developed chronic hepatitis B, titers increased until typical massive viremia was evident. Whereas healthy HBsAg carriers with anti-HBe always had negative genome titers (144 of 144), symptomatic carriers with anti-HBe often had moderate genome titers (9 of 30). It is recommended that genome titers be monitored in HBeAg-positive and in symptomatic anti-HBe positive virus carriers in order to distinguish between virus carriers with high (greater than 5 X 10(7)), moderate (10(5) -5 X 10(7)) and low (less than 10(5)) infectivity.

In a group of 238 homosexual men a hepatitis B vaccine developed at the National Reference Center for Viral Hepatitis (Göttingen/Germany) was evaluated. The vaccine was well tolerated. Three intramuscular injections of inactivated HBsAg (subtype ad; 11300 Natl. Units/dose) induced antibody production in 89.7% of the subjects. Four participants exhibited signs of hepatitis B infection. Vaccine recipients younger than 30 years produced higher antibody levels than older vaccinees. More than one half of the subjects who had only anti-HBs (anti-HBc negative) prior to immunization did not show an anamnestic booster response but formed IgM-anti-HBs. Antibody levels were significantly lower in anti-HBs negative/anti-HBc positive recipients than in subjects who had only anti-HBs or no hepatitis B markers. Immunological studies of T and B cell function did not provide conclusive evidence for the underlying cause of the different immune responses.

The titer of antibody against core antigen of hepatitis B virus in the immunoglobulin M class (IgM anti-HBc) was determined by an IgM capture assay of reduced sensitivity (30 arbitrary units). The distribution of titers among 235 acute hepatitis patients who were hepatitis B surface antigen (HBsAg) positive suggested that 600 U forms a lower cutoff value for acute hepatitis B. Clinically apparent cases of acute hepatitis with high IgM anti-HBc and without HBsAg were rare (2.6%). Acute, non-B hepatitis in HBsAg carriers was more frequent (9.4%). In chronic hepatitis B, 39% of 174 biopsy-proven cases had moderate titers of 30 to 600 U, whereas healthy HBsAg carriers were rarely (4/84) positive. In mild or inapparent infections without HBsAg, titers were between 50 and 400 U. Thus, sufficiently accurate and sensitive quantitation of IgM anti-HBc allows for differentiation of acute and nonacute hepatitis B virus infection in acute hepatitis, partial differentiation between clinically symptomatic and asymptomatic chronic infections, and identification of recent subclinical infections.

Starting with 41.5 l of plasma from anti-HBe positive carriers of HBs antigen, 11,400 doses of a hepatitis B vaccine with 42 micrograms HBsAg-protein and 11,300 national units HBsAg activity per dose were obtained. After purification, HBsAg is obtained in 99% purity with a yield of more than 90% protein. A possible residual infectivity was inactivated by a diluted formalin solution. The infectivity test in chimpanzees confirmed the absence of infectious hepatitis viruses (HBV and nonA-nonB). In guinea pigs the immunogenicity of the vaccine was comparable to that of the reference preparation from the U.S. National Institute of Health. The presence of Al(OH)3 in the vaccine increased the anti-HBs titre by factors of 30-50. After vaccination with two doses 41 of 45 persons became anti-HBs positive, with three doses 42 of 45 persons developed anti-HBs. Median anti-HBs titre after the third doses: schedule I (three doses in intervals of 6 weeks) 427 mWHO-U/ml; schedule II (two doses at an interval of 4 weeks, third doses 4 months after the first doses) 1535 mWHO-U/ml. The vaccine was well tolerated. There were minor local reactions only.

Using recombinant DNA methods, many different approaches may be followed to optimize immunoprophylaxis against hepatitis B viruses. Most obviously a future vaccine should contain besides the known major surface protein two further envelope proteins which have recently been identified. All three envelope proteins should be present on the same particle in natural proportion and conformation. Such a vaccine may induce a more reliable and more durable immune protection even in difficult cases. The protective potential of the viral core protein, in particular of HBeAg, ought to be studied further experimentally. Possibly, the core proteins may be helpful in an immune therapy of already infected persons.

Three groups of subjects (58 females, 54 heterosexual males, and 50 homosexual males) received three doses of a recombinant DNA yeast-derived hepatitis B vaccine according to a 0, 1, and 6 month vaccination schedule. Local and general side effects were mild. Seroconversion rates after three injections were not significantly different between the groups. Females showed a significantly higher anti-HBs response than both groups of males, and heterosexual males had higher antibody titres than homosexual males. Among the four homosexual non-responders, three were carriers of the human immunodeficiency virus.

Within one year four butchers fell ill with acute hepatitis B in a butcher's shop with ten employees. A symptom-free hepatitis B virus carrier was detected among the non-diseased employees. Lack of IgM-anti-HBc antibodies showed that the carrier state had probably existed for more than one year. The time course of infections and the kind of cooperation made it very likely that the carrier had infected his symptomatic mates. The outbreak shows that hepatitis B can be an occupational disease also in non-medical occupations. Should hepatitis B occur in a workman exposed to trauma frequently due to his occupation, serological investigation of the workmates is required in order to identify potential sources of infection and to enable future protective measures.

The frequency of hepatitis B was examined using three serological parameters: HBsAg, antiHBs and antiHBc. All three substances were detected qualitatively by means of sensitive radioimmunological techniques. Of 1216 patients with acute hepatitis, 55 percent were HBsAg and antiHBc positive on admission to hospital. A further 17.8 percent had no HBsAg but were antiHBc positive and also partly antiHBs positive. These cases can be divided into 2 groups: In one group, in 8.6 percent of patients had a high antiHBc concentration during the acute phase. Similar antiHBc concentrations were seldom found (0.04 percent) in HBsAg negative blood donors. AntiHBs in the patients was at first mainly negative and then appeared during reconvalescence. These cases were considered to be acute hepatitis type B, although HBsAg was absent. In the second group, comprising 9.2 percent of the patients, antiHBc was present in low concentrations and in majority of cases antiHBs had been present from the beginning. The same antibody constellation was found in 3.6 percent of 2341 blood donors. In the group of patients it is supposed that the acute hepatitis present is not of type B and has a different aetiology. The low concentration of antibody is interpreted to be a sign of an earlier HBV-infection.

IgM antibody to hepatitis B core antigen (anti-HBc) was determined by a reverse enzyme immunoassay. In all of 58 patients with transient hepatitis B surface antigen (HBsAg)-positive acute hepatitis, IgM anti-HBc was detected in high titer. In 79.3%, IgM anti-HBc disappeared within two years, but in the remaining 12, it was still detectable. In 20 of 21 patients who developed chronic hepatitis, IgM anti-HBc was present after two years. High titers of IgM anti-HBc were found in 13 patients with histologically confirmed hepatitis B in whom HBsAg could not be detected in the initial serum samples; 11 of them later developed antibody to HBsAg and/or e antigen. Furthermore, IgM anti-HGc was detected in eight (5.6%) of 142 HBsAg-negative blood donors with elevated levels of serum transaminases and in 11 (0.5%) of 2,400 HBsAg-negative blood donors with normal levels of serum transaminases. Thus, IgM anti-HBc might be a better indicator of hepatitis B than HBsAg and help differentiage acute from chronic infection in HBsAg-positive patients.

The concentration of HBsAg in serum was determined in arbitrary units by quantitative immunoelectrophoresis and in units of optical density by UV-photometry of purified antigen. The HBsAg was purified from serum by gelchromatography and subsequently by isopycnic centrifugation in cesium chloride. The ratio of the immunoelectrophoretically measured concentration to the units of optical density was found to be constant in six different samples with both subtypes - ad and ay - and varying concentration. The concentration of protein in three purified HBsAg samples was determined after acid hydrolysis by automatic aminoacid analysis. The specific extinction was E1 280 mg/ml = 4,5 +/- 0,3. Thus the arbitrary units of immunoelectrophoresis were converted to concentration of HBsAg specific protein, expressed as mug/ml. In 540 Ausria positive serum samples, mainly from the beginning of an acute hepatitis, 10-40 mug/ml was the most frequent range of concentration. About 4% of the sera contained more than 100 mug/ml, about 20% contained between 0,005 and 0,5 mug/ml and were positive only in RIA. 10 mug/ml correlated with titers in complement fixation of 1:32/64, in counterimmunoelectrophoresis of 1:8 and immuno-diffusion of 1:2. The use of standards with a defined concentration of HBsAg would allow a better control of the sensitivity in qualitative tests and of reproducibility in quantitation.