Immunogenic Domains of HDAg

In the late 1980s and early 1990s some groups investigated the B-cell epitopes of HDV. With the current knowledge about the humoral and cellular immune responses against HDV it has to be stated that the antibody response and, therefore, the corresponding epitopes, do not play an important role in the defense of HDV. Figure 2 gives an overview of the different epitopes characterized by different authors.

Bergmann et al. identified immunogenic epitopes on HDAg by the use of 15 synthetic peptides covering the whole open reading frame (ORF) encoding HDAg (Bergmann et al. 1989). Antisera of humans, chimpanzees, and woodchucks infected with HDV reacted with the peptides. The pattern was distinct for each serum. Many of the sera from all three species recognized sequences from the whole ORF. The unique reactivity profile of each serum could be related to genetic variations among HDV isolates. Residues 52-93 were shown to induce a major immunodominant response in humans by the use of anti-peptide sera in a competition enzyme immunoassay, in immuno-precipitation, and in immunoblotting. Furthermore, in this region a peptide is located which all human sera recognized. Peptides derived from this domain were later used for the vaccination of woodchucks (see Sect. 4.2 and Fig. 2).

In another study 209 overlapping hexapeptides spanning the entire 214 amino acid residues of HDAg were used for the investigation of immunogenic domains on HDAg (Wang et al. 1990). Screening ofthe peptides with five high-titer anti-HDV human sera by ELISA resulted in the identification of seven antigenic domains. Although the carboxy-terminal 50 residues of the HDAg reacted with each of the human sera, evident variation was found between individual sera in respect to the degree to which certain hexapeptides were bound by antibody. To confirm the results three oligopeptides were tested for antigenic activity by microdilution ELISA. Maximal antigenic activity was found with the peptide spanning the residues 156-184, less antigenic activity for peptide 197-211, and only limited activity for peptide 2-17. These results were in concordance with the ELISAs with the hexapeptides (Fig. 2).

On the basis of these results Poisson et al. investigated the antigenic activity of 80 sera of HDV-infected humans with four oligopeptides (Poisson et al. 1993). In agreement with the results of Wang et al., all peptides were recognized; however, the frequency of reactivity was low with peptide 2-17 (Wang et al. 1990). The peptide reacting with most sera (aa 155-172) corresponds to one of the two regions (amino acids 84-111 and aa 155-172) found to be highly conserved between different isolates (Chao et al. 1991). The binding activity of peptide 168-182 was significantly greater than that of the other three peptides. The authors found differences in the immune response to the HDAg-derived peptides between HDV-HBV coinfection or HDV superinfection and also according to the delay between onset of infection and time point of serum sampling (Fig. 2).

In a more recent study a dominant HDAg-specific antibody domain was localized on the N terminus of HDAg (residues 1-83) (Seizer et al. 2005). Balb/c mice were immunized with plasmids encoding three overlapping residues of HDAg fused to the hsp73-binding for efficient expression. Only in mice immunized with the plasmid expressing the N-terminal residues a detectable antibody response (ELISA and western blot) was induced (Fig. 2).

Altogether, four predominant B-cell epitopes have been characterized in the above described studies: residues 2-17, 52-93, 156-184, and 189-211. Bichko et al. showed that three of these domains, corresponding to the nuclear localization signal, the putative assembly domain, and the 19 aa C-terminal extension unique to the L-HDAg, are exposed on HDV. Only the domain between aa 2-17 could not be characterized finally in this study (Bichko et al. 1996).

Patients infected with HDV have been shown to develop antibodies to different antigenic sites of HDAg. However, neutralizing anti-HDAg antibodies which were able to provide protection, have not been found.

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