Immunization with HDV Protein

Prokaryotic and eukaryotic expression systems were used to produce HDV protein for vaccination. Different expression systems result in different protein folding and, therefore, different presentation to the immune system.

Karayiannis et al. studied the immunogenicity of HDAg expressed in E. coli in 1990 (Karayiannis et al. 1990). They immunized four WHV carrier wood-chucks with a fusion protein consisting of the 64 N-terminal amino acids of HDAg and a part of the MS2 polymerase of the bacterial expression vector (pPL31), which proved to be immunogenic in rabbits. This protocol induced a strong humoral immune response in all woodchucks. After challenge the antibody titers raised significantly. However, no protection from infection was observed. The course of infection did not differ from that of the control animals. The HDAg used in this study represented only a part of the total protein. The lack of protection could have been related to the fact that important epitopes for the humoral, or especially the Th and the CTL response, map to the remaining carboxyl end of the protein. Therefore, the same group tested the effect of immunization with the complete S-HDAg expressed in baculovirus using CFA and incomplete Freund's adjuvant (IFA) in one animal (Karayiannis et al. 1993a, 1993b). This immunization protocol did not induce an antibody response and was not protective, but HDV RNA appeared 4-5 weeks later than in the two control animals and was only detectable by nested PCR, not by dot blot hybridization. Postmortem liver biopsies 6 months after challenge revealed the presence of HDAg in the hepatocytes of this animal and, therefore, a chronic HDV infection.

Ponzetto et al. seemed to be more successful using S-HDAg expressed in yeast. They immunized six WHV carrier woodchucks eight times with HDAg without any adjuvant. Two animals seemed to be protected from HDV superinfection. HDV RNA was not detectable by PCR in these animals. Two additional woodchucks presented with viremia of short duration. Unfortunately, the authors did not mention the sensitivity of their tests and the length of the observation period after challenge is missing. This paper is a short communication and has not been published in detail (Ponzetto et al. 1993).

The S-HDAg expressed in yeast was also used for immunization of woodchucks by our group (Fiedler et al. 2001). Four animals were immunized with HDAg with immunostimulatory CpG oligonucleotides in IFA and challenged with HDV later on. HDAg/CpG immunization induced a humoral and a Th-cell response in WHV carrier woodchucks, which was already described in more detail (see Sect. 3.3.3). The HDAg/CpG immunized woodchucks were challenged with 106 genome equivalents of HDV. Neither the humoral nor the Th-cell immune responses were sufficient to protect the woodchucks from HDV superinfection. The course of HDV superinfection was similar to that in woodchucks that were not immunized; namely, the same level of HDV viremia and the typical fluctuation of viremia with characteristic peaks were observed in both groups.

Recently, the same HDAg expressed in yeast was used in another study for vaccination and challenge of eight woodchucks (D'Ugo et al. 2004). Four animals were immunized with HDAg using CFA/IFA as adjuvant, the other four with HDAg using MF59 as adjuvant. MF59 is an oil mineral-water emulsion which has been shown to augment the antigen-specific humoral immune response and to induce a Th-cell response that is more type 2-like in nature (Verschoor et al. 1999). The humoral immune response was detected earlier and at higher titers in the woodchucks immunized with HDAg/CFA vs. those immunized with HDAg/MF59. The Th-cell immune response was already described (see Sect. 3.3.3). After challenge HDV RNA was detected at 2-4 weeks in all animals, indicating that neither of the vaccines was able to protect from superinfection despite the presence of anti-HDV and a Th-cell immune response to HDAg. However, differences were observed in peak serum HDV RNA levels and persistence; namely, the HDAg/CFA immunized animals presented with a delayed appearance and very low HDV RNA titers. Woodchucks in the HDAg/MF59 group showed a trend to survive longer than those in the HDAg/CFA group and the controls. Histological analysis of liver tissues performed before challenge and after death clearly showed that the control animals developed hepatitis-like massive panacinar hepatic necrosis, which was not present in vaccinated woodchucks. In addition, two wood-chucks immunized with HDAg/MF59 had cleared HDV RNA in the livers at autopsy, whereas all other animals still presented with HDV viremia in the liver. The authors speculate that the lack of viremic peak in the HDAg/CFA immunized animals clearly signified an early anti-viral effect, which could have been accompanied by a cell inflammatory response in the liver. In the HDAg/MF59 group the less evident and delayed immune response may have led to an effective anti-viral activity in the absence of an inflammatory response. The type of adjuvant could have influenced the priming of specific T cells.

The combination of HDAg with different adjuvants was able to induce humoral and Th-cell immune responses of different intensities. Protection from superinfection, however, was not achieved. A modulation of the course of infection might have been induced in some, but not all animals.

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