DNA vaccination in combination or not with lamivudine treatment breaks humoral immune tolerance and enhances cccDNA clearance in the duck model of chronic hepatitis B virus infection.

This study used a duck hepatitis B virus (DHBV) model to evaluate whether a novel DNA vaccination protocol alone or associated with antiviral (lamivudine) treatment was able to clear the intrahepatic covalently closed, circular viral DNA (cccDNA) pool responsible for persistence of infection. DHBV carriers received DNA vaccine (on weeks 6, 10, 13, 14, 28 and 35) targeting the large envelope and/or core proteins alone or combined with lamivudine treatment (on weeks 1-8) or lamivudine monotherapy. After 10 months of follow-up, a dramatic decrease in viraemia and liver DHBV cccDNA (below 0.08 cccDNA copies per cell) was observed in 9/30 ducks (30 %) receiving DNA mono- or combination therapy, compared with 0/12 (0 %) from lamivudine monotherapy or the control groups, suggesting a significant antiviral effect of DNA immunization. However, association with the drug did not significantly improve DHBV DNA vaccine efficacy (33 % cccDNA clearance for the combination vs 27 % for DNA monotherapy), probably due to the low antiviral potency of lamivudine in the duck model. Seroconversion to anti-preS was observed in 6/9 (67 %) ducks showing cccDNA clearance, compared with 1/28 (3.6 %) without clearance, suggesting a significant correlation (P<0.001) between humoral response restoration and cccDNA elimination. Importantly, an early (weeks 10-12) drop in viraemia was observed in seroconverted animals, and virus replication did not rebound following the cessation of immunotherapy, indicating a sustained effect. This study provides the first evidence that therapeutic DNA vaccination is able to enhance hepadnaviral cccDNA clearance, which is tightly associated with a break in humoral immune tolerance. These results also highlight the importance of antiviral drug potency and an effective DNA immunization protocol for the design of therapeutic vaccines against chronic hepatitis B.

Sequence-specific inhibition of duck hepatitis B virus reverse transcription by peptide nucleic acids (PNA).

BACKGROUND/AIMS: Peptide nucleic acids (PNAs) appear as promising new antisense agents, that have not yet been examined as hepatitis B virus (HBV) inhibitors. Our aim was to study the ability of PNAs targeting the duck HBV (DHBV) encapsidation signal epsilon to inhibit reverse transcription (RT) and to compare their efficacy with phosphorothioate oligodeoxynucleotides (S-ODNs). METHODS: The effect of two partly overlapping PNAs targeting epsilon and of analogous S-ODNs was tested in cell-free transcription and translation system for DHBV RT expression. In addition their antiviral effect was investigated in primary duck hepatocytes (PDH). RESULTS: Both PNAs reproducibly inhibited DHBV RT in a dose-dependent manner with IC(50) of 10nM, whereas up to 600-fold higher concentration of S-ODNs was required for similar inhibition. The PNA targeting the bulge and upper stem of epsilon appeared as more efficient RT inhibitor than the PNA targeting only the bulge. Importantly, the inhibition was highly sequence-specific since double-mismatched PNA had no effect on the RT reaction. Moreover, in PDH the PNA coupled to Arg(7) cationic delivery peptide decreased DHBV replication. CONCLUSIONS: We provide the first evidence that PNAs targeting the bulge and upper stem of epsilon can efficiently and in a sequence-specific manner inhibit DHBV RT.

Antiviral effect of adefovir in combination with a DNA vaccine in the duck hepatitis B virus infection model.

BACKGROUND/AIMS: Combination of antiviral drugs with immunotherapeutic approaches may be a promising approach for the treatment of chronic hepatitis B. We used the duck HBV (DHBV) infection model to evaluate the efficacy of the combination of adefovir with DNA-immunization by comparison with the respective monotherapies. METHODS: Pekin ducks chronically infected with DHBV received adefovir treatment alone or in association with intramuscular immunization with a plasmid (pCI-preS/S) expressing the DHBV large envelope protein. Ducks immunized with pCI-preS/S plasmid alone and two control groups receiving empty plasmid injections or no treatment were followed in parallel. RESULTS: All animals treated with adefovir showed a marked drop in viremia titers during drug administration, followed by a rebound of viral replication after drug withdrawal. Eight weeks after the third DNA boost, the median of viremia within the duck group receiving the combination therapy tended to be lower compared to that of the other groups. In addition, our results suggest a trend to an additive effect of adefovir and DNA vaccine since a 51% decrease in DHBV DNA was observed in autopsy liver samples from combination therapy group, whereas pCI-preS/S or adefovir monotherapies decreased intrahepatic viral DNA by 38 and 14%, respectively. This effect was sustained since it was observed 12 weeks after the end of therapy. CONCLUSIONS: Our results suggest that combination of adefovir with DNA-vaccine may be able to induce a sustained antiviral effect in vivo.

Progress in DNA vaccine for prophylaxis and therapy of hepatitis B.

Increasing lines of evidence suggest that DNA vaccine is of interest to fight chronic hepatitis B virus (HBV) infection. We used the Pekin duck infected by duck HBV (DHBV), closely related to the human virus, which is an attractive model allowing study of protective and therapeutic effectiveness of DNA vaccines against hepatitis B. Immunisation with a plasmid encoding the DHBV large (L) envelope protein induced a strong, specific, highly neutralising and long-lasting anti-preS humoral response in uninfected ducks. Importantly, maternal antibodies elicited by such DNA immunisation were vertically transmitted and protected progeny against viral challenge. Therapeutic immunisation of chronic DHBV-carrier ducks with this plasmid DNA led to the dramatic and sustained decrease in viral replication and even to clearance of intrahepatic viral covalently close circular DNA (cccDNA) pool in some animals. Our recent combination therapy data showed even a more pronounced antiviral effect of DNA vaccine to DHBV envelope protein when associated with antiviral drug (lamivudine) treatment. Therefore, DNA-based vaccine appears as a promising new approach for prophylaxis and therapy of hepatitis B.