Tanja Bauer

High genetic variability of the group-specific a-determinant of hepatitis B virus surface antigen (HBsAg) and the corresponding fragment of the viral polymerase in chronic virus carriers lacking detectable HBsAg in serum

Chronic carriers of hepatitis B virus (HBV) usually show hepatitis B surface antigen (HBsAg) in their sera, which is considered the best marker for acute and chronic HBV infection. In some individuals, however, this antigen cannot be detected by routine serological assays despite the presence of virus in liver and peripheral blood. One reason for this lack of HBsAg might be mutations in the part of the molecule recognized by specific antibodies. To test this hypothesis, the HBV S gene sequences were determined of isolates from 33 virus carriers who were negative for HBsAg but showed antibodies against the virus core (anti-HBc) as the only serological marker of hepatitis B. Isolates from 36 HBsAg-positive patients served as controls. In both groups, a considerable number of novel mutations were found. In isolates from individuals with anti-HBc reactivity only, the variability of the major hydrophilic loop of HBsAg, the main target for neutralizing and diagnostic antibodies, was raised significantly when compared with the residual protein (22. 6 vs 9.4 mutations per 1000 amino acids; P<0.001) and with the corresponding region in the controls (22.6 vs 7.5 exchanges per 1000 residues; P<0.001). A similar hypervariable spot was identified in the reverse transcriptase domain of the viral polymerase, encoded by the same nucleotide sequence in an overlapping reading frame. These findings suggest that at least some of the chronic low-level carriers of HBV, where surface antigen is not detected, could be infected by diagnostic escape mutants and/or by variants with impaired replication.

A novel deletion mutant of hepatitis B virus surface antigen

HBsAg is the most important serological marker for acute or chronic hepatitis B. Nevertheless, there are reports of HBsAg‐negative virus carriers, either with anti‐HBc as the only marker for hepatitis B virus (HBV) infection or even positive for anti‐HBs and anti‐HBc. We report isolates from a patient, in which a deletion in the HBs‐gene was associated with persisting viremia in the presence of anti‐HBs. The 62‐year‐old female, infected most likely by her husband, had detectable markers of chronic active hepatitis B, such as HBsAg, HBeAg, and anti‐HBc‐IgM, for 2 years. The patient then seroconverted to anti‐HBs, although HBeAg and anti‐HBc‐IgM remained detectable. At this time, semiquantitative polymerase chain reaction showed about 104 viral genomes per milliliter of serum. Direct sequencing of the amplified products revealed a major population of DNA molecules with a deletion of nucleotide 31 of the HBs‐gene, which up to now has not been described. This deletion led to a frame‐shift and introduced a stop‐codon after 21 amino acids of the sHBsAg. We suspect that this deletion, and the resulting HBsAg lacking the major epitopes recognized by specific antibodies, could favor ongoing viral replication, despite the presence of anti‐HBs. However, because the reading frame of the polymerase was also severely damaged by this deletion, it is assumed that a minor population of intact genomes was present to help in the formation of virus particles. J. Med. Virol. 58:105–110, 1999.

Immunization with an adjuvant hepatitis B vaccine in liver transplant recipients: Antibody decline and booster vaccination with conventional vaccine

Patients after orthotopic liver transplantation (OLT) due to hepatitis B virus (HBV)‐related disease are at risk of endogenous hepatitis B reinfection and may receive life long prophylaxis with hepatitis B hyperimmunoglobulin (HBIG). In a previous study 16 of 20 OLT patients were immunized successfully with an adjuvant hepatitis B vaccine. To maintain protective antibody levels under immunosuppressive therapy, 11 of these patients were revaccinated with a double dosed conventional hepatitis B vaccine. Median interval between last vaccination and booster was 24 months (range 22‐31 months). Antibody titres against hepatitis B surface antigen (anti‐HBs) were monitored at the day of booster vaccination (day 0), at day 7 and day 28. At day 0, all vaccinees but one had anti‐HBs titres greater than 500 IU/L (median 1,925 IU/L, range 196‐7,612 IU/L). Maximum antibody titres after previous vaccination declined by a median of 82% (range 47‐96%). After booster vaccination the anti‐HBs titre increased significantly by a median factor of 2.42 (P<0.05). In conclusion, the majority of liver transplant recipients who previously had responded to adjuvant hepatitis B vaccine exhibited sufficient immunocompetence to produce a substantial antibody response after booster immunization with a conventional vaccine. Liver Transpl 12:316–319, 2006.

Vaccination against hepatitis B in liver transplant recipients : Pilot analysis of cellular immune response shows evidence of HBsAg-Specific regulatory T cells

After liver transplantation for hepatitis‐B‐related diseases, patients currently receive lifelong treatment with hepatitis B immunoglobulin to prevent endogenous reinfection with hepatitis B virus (HBV). Active immunization with hepatitis B vaccine would be a preferable alternative; however, most attempts to immunize these patients with standard vaccine have failed. A recent study with a new adjuvanted hepatitis B vaccine was exceptionally successful, leading to a high‐titered long‐lasting antibody response in 80% of all vaccinees. To identify the immunological mechanisms behind these unexpected results, the successfully vaccinated participants were tested for hepatitis B surface antigen (HBsAg)‐specific T and B cells, and their cellular responses to revaccination with conventional vaccine were studied. HBsAg‐specific CD4+ T lymphocytes could be detected in 13 of 16 patients after immunization with the new vaccine. Unexpectedly, these T cells produced almost exclusively interleukin (IL)‐10 and had a CD4+/CD25+ phenotype. They were functionally active, suppressing cytokine secretion in HBsAg‐specific (Th1) cells, thus representing antigen‐specific regulatory T cells (TReg). Following a booster dose with conventional vaccine 22‐31 months after completion of the initial vaccination series, the T‐cell pattern in the revaccinated individuals changed substantially: 7 days after revaccination 9 of 11 individuals showed a switch to a Th1‐type immune response with HBsAg‐specific T cells secreting IL‐2, interferon gamma and tumor necrosis factor alpha as observed in healthy controls. Four weeks after the booster, 4 patients still showed a Th1‐type cytokine pattern, whereas in 5 patients only IL‐10‐secreting cells were detectable. After 1 year, in 3 of 4 revaccinated individuals only IL‐10‐secreting cells could be found, whereas the specific T cells of the fourth patient still showed a Th1‐type of response. HBsAg‐specific TReg cells could be demonstrated in HBV‐positive liver transplant recipients successfully immunized with a new adjuvanted vaccine. Revaccination led to immediate disappearance of the these cells and the appearance of HBsAg‐specific T cells with a Th1‐type cytokine profile, which in most cases were replaced by the IL‐10‐secreting regulatory cells during the following months. The specific induction of TReg cells could contribute to the poor response of liver transplant recipients to conventional vaccine. In conclusion,, for successful vaccination of these patients, a vaccine with a strong inhibitory effect on TReg cells would be desirable. Liver Transpl 13:434–442, 2007.

Urea-mediated cross-presentation of soluble Epstein-Barr virus BZLF1 protein.

Soluble extracellular proteins usually do not enter the endogenous human leukocyte antigen (HLA) I–dependent presentation pathway of antigen-presenting cells, strictly impeding their applicability for the re-stimulation of protein-specific CD8+ cytotoxic T lymphocytes (CTL). Here we present for the Epstein-Barr virus (EBV) BZLF1 a novel strategy that facilitates protein translocation into antigen-presenting cells by its solubilisation in high molar urea and subsequent pulsing of cells in presence of low molar urea. Stimulation of PBMC from HLA-matched EBV-seropositive individuals with urea-treated BZLF1 but not untreated BZLF1 induces an efficient reactivation of BZLF1-specific CTL. Urea-treated BZLF1 (uBZLF1) enters antigen-presenting cells in a temperature-dependent manner by clathrin-mediated endocytosis and is processed by the proteasome into peptides that are bound to nascent HLA I molecules. Dendritic cells and monocytes but also B cells can cross-present uBZLF1 in vitro. The strategy described here has potential for use in the development of improved technologies for the monitoring of protein-specific CTL.

Two decades of hepatitis B vaccination in mentally retarded patients: effectiveness, antibody persistence and duration of immune memory.

INTRODUCTION Institutionalized mentally retarded subjects are well-known to be at-risk for HBV infection. We studied the persistence of vaccine-induced anti-HBs antibodies and the robustness of the HBsAg-specific immune memory in this population, 18-20 years after the first vaccine dose. MATERIALS AND METHODS Non-immune residents of 4 institutions were immunized in 1984-1986. In 2004, 207 subjects were bled to determine humoral and cellular immune memory. Immune response to a booster dose was evaluated in subjects with anti-HBs level <100 IU/L. RESULTS Four subjects showed anti-HBc seroconversion, without clinical implications. Pre-booster anti-HBs levels <100 IU/L were found in 45 subjects (22%); 34/39 (87%) responded with a rapid and high anti-HBs titer to the booster dose. Robust T and B cell memory was present pre- and post-booster. DISCUSSION AND CONCLUSION Overall results confirm that hepatitis B vaccines are highly effective and immunogenic, and confer long-term persistence of antibodies and immune memory in an at-risk population.