Klaus-Hinrich Heermann

Identification of an attachment site for human liver plasma membranes on hepatitis B virus particles

The surface antigen of hepatitis B virus (HBsAg) exposes three protein domains: preS1, preS2, and S. In a previous study we have shown that preS1 sequences expressed in transfected yeast cells bind specifically to plasma membranes of human liver. In this study we show that purified virus particles from a virus carrier bind also specifically to such membranes. Subviral HBsAg filaments which are rich in preS1 bind well too, while HBsAg 20-nm particles which contain small amounts of preS1 bind to a much lesser degree. The binding can be inhibited by a monoclonal antibody which recognizes a sequential epitope between amino acids 27 and 49 of the preS1 domain.

Immunogenicity of the Gene S and Pre-S Domains in Hepatitis B Virions and HBsAg Filaments

The three morphological forms (20-nm particles, filaments, virions) of hepatitis B surface antigen (HBsAg) were isolated from serum of chronic virus carriers or from transfected cell lines. BALB/c mice and guinea pigs were immunized with the antigens and the antibody responses against the three antigenic domains of the viral envelope were assayed. The proportion of pre-S1, pre-S2 and gene S antibodies was similar to the molar proportion of the domains in the immunogens. The major gene S and pre-S1 epitopes were conformational, and the major epitopes of the pre-S2 domain were sequential. The immunogenicity of natural and recombinant antigens was identical. The proportion of subtype-specific antibodies was high. The results suggest that recombinant HBsAg filaments containing both subtypes ad and ay may be optimal hepatitis B vaccines.

Mapping of immunodominant B-cell epitopes and the human serum albumin-binding site in natural hepatitis B virus surface antigen of defined genosubtype

Twelve MAbs were generated by immunization of BALB/c mice with plasma-derived hepatitis B virus surface spherical antigen particles subtype ayw2 (HBsAg/ayw2 genotype D). Their epitopes were mapped by analysis of reactivity with plasma-derived HBsAg/ayw2 and HBsAg/adw2 (genotype A) in enzyme immunoassays and blots. Mapping was supported by nested sets of truncated preS2 proteins and preS2 peptides. Five antibodies were S domain-specific, seven were preS2-specific and 11 had a preference for genotype D. According to our data, group I of the three known epitope groups of preS2 has to be divided into IA and IB. Three preS2-specific MAbs forming the new group IA reacted with genotype D residues 3-15 which have not yet been described as an epitope region. IA antibodies strongly inhibited the binding of polymerized human serum albumin. Two antibodies (group II) reacted with the glycosylated N-terminal region of preS2 in plasma-derived HBsAg, but not with a preparation from transfected murine cells. One group III antibody was subtype-specific and reacted with the highly variable preS2 sequence 38-48. Only one antibody (group IB) mapped to the region (old group I) which was believed to be immunodominant and genotype-independent. Geno(sub)type-specific epitopes of preS2 are obviously the immunodominant components of natural HBsAg in BALB/c mice, but these epitopes may be masked by serum albumins in humans. The data may explain why it is difficult to detect anti-preS2 antibodies in human recipients of preS2-containing vaccines, in spite of the preS2 immunodominance in mice.

Testing of individual blood donations for HCV RNA reduces the residual risk of transfusion-transmitted HCV infection.

BACKGROUND: To allow cost‐effective RNA testing with NAT techniques, the national authorities of several countries have planned or already introduced tests of mixed specimens, that is, plasma pools.

Nature and display of hepatitis B virus envelope proteins and the humoral immune response.

ConclusionsThe pattern of display of envelope proteins on HBV indicates that the preS domains have an important role in virus assembly and secretion. Furthermore sites of interaction with the membrane of hepatocytes and of other cells which are susceptible to infection have been identified within the preS1 and preS2 protein domains. These observations support the concept that these sequences represent the virus attachment sites for the host cell membrane. The nature of the cellular receptors involved in this binding has not yet been fully clarified. Several B and T cell epitopes, which evoke antibody and cellular response in humans, have been identified within the preS1 and preS2 protein domains. The immune response to some of these determinants seems to be particularly relevant in virus neutralization and in termination of virus replication. Their exact characterization is needed for possible inclusion in recombinant HB vaccines, to further increase their efficacy. The immune response to preS may also be involved in the pathogenesis of liver disease, as preS1 and preS2 epitopes are recognized by cytotoxic T lymphocytes during chronic infection and after HB vaccination. These findings need to be extended further and may provide new important information on the pathogenesis of HBV infection.

Genomic variability in the preS1 region and determination of routes of transmission of hepatitis B virus

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.

Low Prevalence of Hepatitis C Virus Antibodies and RNA in Patients with Myocarditis and Dilated Cardiomyopathy

We investigated the prevalence of hepatitis C virus in patients with dilated cardiomyopathy (DCM) and myocarditis in comparison to a control group of patients suffering from noninflammatory cardiac diseases such as aortic stenosis. In contrast to the results of previous studies on small numbers of patients, no significant difference in the prevalence of hepatitis C infections was observed. Our data suggest that HCV is not an important causal agent for myocarditis and DCM.

High-throughput extraction, amplification, and detection (HEAD) of HCV-RNA in individual blood donations

BACKGROUND High-throughput nucleic acid amplification techniques (NATs) are required for the detection of viral genomes in individual blood donations and might be helpful in any virological laboratory. OBJECTIVE To develop and automate a method for the detection of hepatitis C virus RNA in individual blood donations, compatible with the time schedule of routine blood bank screening an product release. STUDY DESIGN The viral RNA was isolated with the use of target specific capture oligonucleotides and magnetic beads. This extraction method was combined with reverse transcription/amplification (RT/PCR) and fluorescence detection. We adapted our method on a pipetting robot and pipetted all steps in a single room. When the pipetting was completed, microtiter plates were heat-sealed with foils and placed into a thermocycler. Positive reactions were detected with a fluorescent dye in a second room. Aerosols were avoided with programmed slow pipetting steps and with a special device constructed for the removal of the used disposable tips. During a 7 month period, we used this method in routine testing of individual donations prior to the release of all blood components. RESULTS The total number of 11,700 individual donations including platelet concentrates were analysed. We tested up to 192 specimens in one run within 7 h. The frequency of cross-contamination using the automated procedure was 0.1%. Five specimens have been found repeatedly reactive for HCV-RNA, four of these were anti-HCV positive, one sample from a repeat donor was negative in anti-HCV assays. A seroconversion was detectable at his next presentation, 6 months later. CONCLUSION In this pilot study, we demonstrate that automated HCV-RT-PCR testing is practicable for individual donations in high-throughput. Additionally, the described PCR approach could easily be adapted to the detection of other viral genomes by the use of specific primers.

Specific magnetic bead-based capture of free fetal DNA from maternal plasma.

An automated magnetic capture hybridization (MCH) method for the extraction and enrichment of fetal RHD specific DNA fragments from maternal plasma was developed using plasma from 1000 D-negative pregnant women. A real time PCR protocol for RHD exon 7 was applied. MCH was compared with the QIAamp DSP Virus Kit (QIAamp) as a reference. Compared with the QIAamp method, the percentage of fetal DNA increased from 2.86% to 4.83% (p<0.05, n=8). The 95% detection limit of MCH was determined at 286 pg/ml (43 geg/ml) compared with 138 pg/ml (21 geq/ml) for the QIAamp DSP Virus Kit.

Fetal DNA: strategies for optimal recovery.

For fetal DNA extraction, in principle each DNA extraction method can be used; however, because most methods have been optimized for genomic DNA from leucocytes, we describe here the methods that have been optimized for the extraction of fetal DNA from maternal plasma and validated for this purpose in our laboratories. The use of the QIAamp DSP Virus kit (QIAGEN), the QIAamp DNA Blood Mini kit (QIAGEN), and the Magna Pure LC (Roche) is based on the kit components provided by the respective companies. However, we noticed that the yield of fetal DNA from maternal plasma can be increased when higher volumes are processed or some slight modifications of the protocols provided by the manufacturer are followed. Here, we also describe an in-house method that allows the specific capture of target molecules in an extremely low volume by using magnetic beads and magnetic tips. This method can be either performed by hand, or it can be adapted to a commercially available pipetting workstation.