In vivo infectivity of liver extracts after resolution of hepadnaviral infection following therapy associating DNA vaccine and cytokine genes.

DNA-based vaccination appears of promise for chronic hepatitis B immunotherapy, although there is an urgent need to increase its efficacy. In this preclinical study, we evaluated the therapeutic benefit of cytokine (IL-2, IFN-γ) genes co-delivery with DNA vaccine targeting hepadnaviral proteins in the chronic duck hepatitis B virus (DHBV) infection model. Then, we investigated the persistence of replication-competent virus in the livers of apparently resolved animals. DHBV carriers received four injections of plasmids encoding DHBV envelope and core alone or co-delivered with duck IL-2 (DuIL-2) or duck IFN-γ (DuIFN-γ) plasmids. After long-term (8 months) follow-up, viral covalently closed circular (ccc) DNA was analysed in duck necropsy liver samples. Liver homogenates were also tested for in vivo infectivity in neonatal ducklings. Co-delivery of DuIFN-γ resulted in significantly lower mean viremia starting from week 21. Viral cccDNA was undetectable by conventional methods in the livers of 25% and 57% of animals co-immunized with DuIL-2 and DuIFN-γ, respectively. Interestingly, inoculation of liver homogenates from 7 such apparently resolved animals, exhibiting cccDNA undetectable in Southern blotting and DHBV expression undetectable or restricted to few hepatocytes, revealed that three liver homogenates transmitted high-titre viremia (3-5×10(10) vge/mL) to naïve animals. In conclusion, our results indicate that IFN-γ gene co-delivery considerably enhances immunotherapeutic efficacy of DNA vaccine targeting hepadnaviral proteins. Importantly, we also showed that livers exhibiting only minute amounts of hepadnaviral cccDNA could induce extremely high-titre infection, highlighting the caution that should be taken in occult hepatitis B patients to prevent HBV transmission in liver transplantation context.

Combination of an antiviral drug and immunomodulation against hepadnaviral infection in the woodchuck model.

The essential role of multispecific immune responses for the control of hepatitis B virus (HBV) infection implies the need of multimodal therapeutic strategies for chronic HBV infection, including antiviral chemotherapy and immunomodulation. This hypothesis was tested in the woodchuck model by a combination of lamivudine pretreatment and subsequent immunizations of woodchucks chronically infected with woodchuck hepatitis virus. The immunizations were performed with DNA vaccines or antigen-antibody immune complexes (IC)/DNA vaccines. Immunizations with IC/DNA vaccines led to an anti-woodchuck hepatitis virus surface antibody response and significant reductions of viral load and antigenemia, suggesting that such a strategy may be effective against chronic HBV infection.

Inhibition of hepadnaviral replication by polyethylenimine-based intravenous delivery of antisense phosphodiester oligodeoxynucleotides to the liver.

Antisense oligodeoxynucleotides (ODNs) appear as attractive anti-hepatitis B virus (HBV) agents. We investigated in vivo, in the duck HBV (DHBV) infection model, whether linear polyethylenimine (lPEI)-based intravenous delivery of the natural antisense phosphodiester ODNs (O-ODNs) can prevent their degradation and allow viral replication inhibition in the liver. DHBV-infected Pekin ducklings were injected with antisense O-ODNs covering the initiation codon of the DHBV large envelope protein, either in free form (O-ODN-AS2) or coupled to lPEI (lPEI/O-ODN-AS2). Following optimization of lPEI/O-ODN complex formulation, complete O-ODN condensation into a homogenous population of small (20-60 nm) spherical particles was achieved. Flow cytometry analysis showed that lPEI-mediated transfer allowed the intrahepatic delivery of lPEI/O-ODN-AS2 to increase three-fold as compared with the O-ODN-AS2. Following 9-day therapy the intrahepatic levels of both DHBV DNA and RNA were significantly decreased in the lPEI/O-ODN-AS2-treated group as compared with the O-ODN-AS2-treated, control lPEI/O-ODN-treated, and untreated controls. In addition, inhibition of intrahepatic viral replication by lPEI/O-ODN-AS2 was not associated with toxicity and was comparable with that induced by the phosphorothioate S-ODN-AS2 at a five-fold higher dose. Taken together, our results demonstrate that phosphodiester antisense lPEI/O-ODN complexes specifically inhibit hepadnaviral replication. Therefore we provide here the first in vivo evidence that intravenous treatment with antisense phosphodiester ODNs coupled to lPEI can selectively block a viral disease-causing gene in the liver.

[Anti-virus effect of combination of attenuated measles virus with 3TC on duck hepatitis B model].

OBJECTIVE: To evaluate the anti-virus effect of combination of attenuated measles virus with 3TC on duck hepatitis B model. METHODS: Guanzhou brown spot ducklings infected with DHBV were divided into three groups. Drug treatments including measles vaccine, 3TC and both measles vaccine and 3TC were given in the 13th day after the infection, respectively. Serum samples were obtained from all ducklings before, during and after treatment at different times and stored at -70 degrees C. DHBV DNA was detected by Dot-blot hybridization. RESULTS: Compared with the levels of DHBV DNA before and after treatment, no statistic difference was found in group of 3TC alone. The levels of DHBV DNA were not changed in group of measles vaccine alone in early stage, but decrease of DHBV DNA occurred after stopping treatment (comparison of the levels T13 with before treatment and T13 of positive group, P < 0.01 and P < 0.05, respectively). DHBV DNA level in group of measles vaccine combined with 3TC was obviously lower at 5th day after the treatment than in both measles vaccine and 3TC alone groups at the same time(P < 0.05 and P < 0.01, respectively), and it remained low when the treatment was discontinued. CONCLUSION: Attenuated measles virus combined with 3TC may have early and long-lasting inhibiting effect on the replication of DHBV.

Recombinant vaccinia virus expressing Pre-S/S protein of duck hepatitis B virus and its preliminary use for treatment of persistent infection.

The envelope (Pre-S/S) gene of duck hepatitis B virus (DHBV) was amplified by polymerase chain reaction (PCR) and cloned into plasmid pGJP5, under the control of vaccinia virus promoter P(7.5). By recombination in cell culture, and screened in human TK- 143 cells in the presence of 5-bromouracil deoxyriboside (5-BUdR), a recombinant vaccinia virus, bearing the envelope gene of DHBV (pGDHBV-5) which could replicate in cell cultures was constructed. DHBV surface antigen (DHBsAg) was detected in pGDHBV-5-infected cell lysate by dot enzyme immunoassay (EIA). After multiple-site intradermal injections of pGDHBV-5, DHBsAg could be detected in the serum of immunized adult ducks. This indicated that the recombinant virus replicated and expresed DHBsAg in ducks. The recombinant virus was used as a therapeutic vaccine to immunize persistently DHBV-infected ducks. After immunization, a transient significant decrease of serum DHBsAg was observed.

Therapy of hepadnavirus infection using antisense oligonucleotides.

Chronic infection with the hepatitis B virus (HBV) is a major health problem worldwide. The only established therapy is alpha-interferon with an efficacy of only 30-40% in highly selected patients. Major theoretical problems of therapeutical strategies against hepadnaviral infections are the limited immune response and the presence of covalently closed HBV DNA in the nucleus. Many nucleoside analogues and inhibitors of viral reverse transcriptases were tested in vitro and in vivo with transient effects and often severe side effects. Molecular therapeutic strategies include antisense DNA/RNA and ribozymes. In vitro antisense oligodeoxynucleotides could be shown to inhibit viral replication and gene expression in human hepatoma cell lines. In vivo an antisense oligodeoxynucleotide directed against the 5'-region of the preS gene of the duck hepatitis B virus inhibited the viral replication and gene expression in ducks. These results demonstrate the potential clinical use of antisense DNA/RNA as antiviral therapeutics.

Immunological responsiveness of Pekin ducks to core antigen of duck hepatitis B virus.

Cellular immune responses to the HBc or HBe antigen of hepatitis B viruses contribute to the pathogenesis of hepatitis B and to the elimination of the virus. Insufficient cytotoxic immune reactions against the core antigen may be one major reason for viral persistence in the absence of severe clinical symptoms. We attempted to stop viral persistence in the animal model of congenitally infected ducks by injection of recombinant DHBc particles, together with the strong immunostimulator Freund's adjuvant. However, the duck HBc antigen (DHBcAg)-treated ducks did not develop detectable liver disease, nor was the virus persistence affected. The congenitally infected ducks did not contain antibodies against DHBcAg before injection despite continuous production of duck hepatitis B virus, and developed only a weak transient antibody response after injection of recombinant DHBcAg together with Freund's adjuvant. Noninfected ducks developed, in contrast, a strong antibody response to the injected DHBcAg. We conclude that congenitally infected ducks are immunotolerant to DHBcAg and cannot be cured by immunotherapy with exogenous recombinant DHBcAg.

In vivo antiviral effects of mismatched double-stranded RNA on duck hepatitis B virus.

The antiviral activity and ability of mismatched double-stranded RNA (m-dsRNA), r(I)n.r(C12-U)n, to induce interferon (IFN) were evaluated in ducks chronically infected with duck hepatitis B virus (DHBV). When m-dsRNA was administered intravenously at a single dose of 5 mg/kg, serum DHBV DNA concentrations decreased significantly for 3 days (P < 0.002). However, the DHBV DNA concentrations returned to the pretreatment levels 4 days after treatment. Inhibition of DHBV DNA replication in the liver was also observed 2 days after treatment. Serum IFN activity peaked 3 hours after administration of m-dsRNA, then rapidly declined. 2'-5' Oligo-adenylate synthetase (2'-5'AS) activity increased gradually after treatment and remained elevated for at least 48 hours. In ducks receiving m-dsRNA once daily for 7 consecutive days, serum DHBV DNA concentrations on the last day of treatment were decreased by 76 +/- 12% (P < 0.05) in ducks that received 0.2 mg of m-dsRNA per kg and by 65 +/- 12% (P < 0.05) in ducks that received 1 mg of m-dsRNA per kg. This decrease persisted for at least 2 weeks after the cessation of treatment in all ducks. These results suggest that m-dsRNA effectively inhibits DHBV replication in vivo, and that IFN induction and stimulation of 2'-5'AS activity contribute to the inhibition of DHBV replication by m-dsRNA.

Solid matrix-antibody-antigen complex can clear viraemia and antigenaemia in persistent duck hepatitis B virus infection.

One-day-old ducklings experimentally infected with duck hepatitis B virus (DHBV) were found to be immunologically tolerant to virus antigens (DHBsAg, DHBcAg), with no humoral or cellular immune responses being detected. When immunized with virus antigens in Freund's complete adjuvant, no immune responses could be induced. Rabbit anti-DHBs sera were complexed to a solid matrix (Staphylococcus aureus Cowan A strain) and purified DHBsAg was bound to this complex to form a solid matrix antibody-antigen (SMAA) complex. This SMAA was used as an immunogen to immunize the tolerant ducks. After three injections, in 12 of 17 ducks serum DHBV DNA became absent and serum DHBsAg was cleared. In eight of 16 ducks, low titres of anti-DHBs could be detected. The SMAA approach shows potential for application in immunoregulatory treatment for chronically infected hepatitis B patients.