Tracing the evolutionary history of hepadnaviruses in terms of e antigen and middle envelope protein expression or processing.

Hepatitis B virus (HBV) is the prototype of hepadnaviruses, which can be subgrouped into orthohepadnaviruses infecting mammals, avihehepadnaviruses of birds, metahepadnaviruses of fish, and herpetohepadnaviruses of amphibians and reptiles. The middle (M) envelope protein and e antigen are new additions in the evolution of hepadnaviruses. They are alternative translation products of the transcripts for small (S) envelope and core proteins, respectively. For HBV, e antigen is converted from precore/core protein by removal of N-terminal signal peptide followed by furin-mediated cleavage of the basic C-terminus. This study compared old and newly discovered hepadnaviruses for their envelope protein and e antigen expression or processing. The S protein of bat hepatitis B virus (BHBV) and two metahepadnaviruses is probably myristoylated, in addition to two avihepadnaviruses. While most orthohepadnaviruses express a functional M protein with N-linked glycosylation near the amino-terminus, most metahepadnaviruses and herpetohepadnaviruses probably do not. These viruses and one orthohepadnavirus, the shrew hepatitis B virus, lack an open precore region required for e antigen expression. Potential furin cleavage sites (RXXR sequence) can be found in e antigen precursors of orthohepadnaviruses and avihepadnaviruses. Despite much larger precore/core proteins of avihepadnaviruses and their limited sequence homology with those of orthohepadnaviruses, their proximal RXXR motif can be aligned with a distal RXXR motif for orthohepadnaviruses. Thus, furin or another basic endopeptidase is probably the shared enzyme for hepadnaviral e antigen maturation. A precore-derived cysteine residue is involved in forming intramolecular disulfide bond of HBV e antigen to prevent particle formation, and such a cysteine residue is conserved for both orthohepadnaviruses and avihepadnaviruses. All orthohepadnaviruses have an X gene, while all avihepadnaviruses can express the e antigen. M protein expression appears to be the most recent event in the evolution of hepadnaviruses.

Molecular cloning, characterization and expression analysis of Tim-3 and Galectin-9 in the woodchuck model.

In recent years, a critical role for T cell immunoglobulin mucin domain 3 (Tim-3) and its ligand Galectin-9 (Gal-9) has emerged in infectious disease, autoimmunity and cancer. Manipulating this immune checkpoint may have immunotherapeutic potential and could represent an alternative approach for improving immune responses to viral infections and cancer. The woodchuck (Marmot monax) infected by woodchuck hepatitis virus (WHV) represents an informative animal model to study HBV infection and HCC. In the current study, the cDNA sequences of woodchuck Tim-3 and Gal-9 were cloned, sequenced and characterized. The extracellular domain of Tim-3 cDNA sequence consisted of 576bp coding sequence (CDS) that encoded 192 amino acids. The 1076bp full-length Gal-9 cDNA sequence consisted of 1059bp coding sequence (CDS) that encoded 352 amino acids with a molecular weight of 39.7kDa. The phylogenetic tree analysis revealed that the woodchuck Tim-3 and Gal-9 had the closest genetic relationship with Ictidomys tridecemlineatus. The result of quantification PCR analysis showed that ubiquitous expression of Gal-9 but not Tim-3 in different tissues of naive woodchucks. Elevated liver Gal-9 expression was observed in woodchucks with chronic WHV infection. Moreover, a polyclonal antibody against the extracellular domain of woodchuck Tim-3 were generated and identified by flow cytometry. Our results serve as a foundation for further insight into the role of Tim-3/Galectin-9 signaling pathway in viral hepatitis and HCC in the woodchuck model.

Nucleoside analogues alone or combined with vaccination prevent hepadnavirus viremia and induce protective immunity: alternative strategy for hepatitis B virus post-exposure prophylaxis.

OBJECTIVES: The current strategies for hepatitis B virus (HBV) post-exposure prophylaxis (PEP) are not generally available in remote and rural areas of developing countries and/or carry potential risks for infection with blood-borne transmitted pathogens. Nucleotide analogues (NAs) are successfully used for human immunodeficiency virus PEP, and maybe effective for HBV PEP. In this study, we tested the NA-based strategies for HBV PEP using the Chinese woodchuck model. METHODS: Chinese woodchucks were inoculated intravenously with different doses of woodchuck hepatitis virus (WHV). A deoxyguanosine analogue entacavir (ETV), a DNA vaccine pWHcIm, or ETV plus pWHcIm were applied to the infected animals 24h later. Twenty weeks later, the animals were re-challenged with WHV to test for the presence of immunity against WHV. RESULTS: Inoculation with different WHV doses had a strong influence on the course of WHV infection; NA alone or in combination with a DNA vaccine completely prevented viremia after a high dose of WHV inoculation in Chinese woodchucks and induced partial or complete protective immunity, respectively. CONCLUSIONS: NA-based PEP strategies (NA alone or in combination with vaccine) may be an alternative of HBV PEP, especially in those living in the remote and rural areas of the developing countries and the non-responders to the current vaccine, and may be valuable in the PEP of HBV and HIV co-infection after occupational and non-occupational exposure. Further clinical studies are warranted to confirm the valuable of NA-based strategies in HBV PEP.

In vivo electroporation improves therapeutic potency of a DNA vaccine targeting hepadnaviral proteins.

This preclinical study investigated the therapeutic efficacy of electroporation (EP)-based delivery of plasmid DNA (pDNA) encoding viral proteins (envelope, core) and IFN-γ in the duck model of chronic hepatitis B virus (DHBV) infection. Importantly, only DNA EP-therapy resulted in a significant decrease in mean viremia titers and in intrahepatic covalently closed circular DNA (cccDNA) levels in chronic DHBV-carrier animals, compared with standard needle pDNA injection (SI). In addition, DNA EP-therapy stimulated in all virus-carriers a humoral response to DHBV preS protein, recognizing a broader range of major antigenic regions, including neutralizing epitopes, compared with SI. DNA EP-therapy led also to significant higher intrahepatic IFN-γ RNA levels in DHBV-carriers compared to other groups, in the absence of adverse effects. We provide the first evidence on DNA EP-therapy benefit in terms of hepadnaviral infection clearance and break of immune tolerance in virus-carriers, supporting its clinical application for chronic hepatitis B.

Enhanced magnitude and breadth of neutralizing humoral response to a DNA vaccine targeting the DHBV envelope protein delivered by in vivo electroporation.

We explored in the duck hepatitis B virus (DHBV) model the impact of electroporation (EP)-mediated DNA vaccine delivery on the neutralizing humoral response to viral preS/S large envelope protein. EP enhanced the kinetics and magnitude of anti-preS response compared to the standard needle DNA injection (SI). Importantly, EP dramatically enhanced the neutralizing potency of the humoral response, since antibodies induced by low DNA dose (10 µg) were able to highly neutralize DHBV and to recognize ten antigenic regions, including four neutralization epitopes. Whereas, SI-induced antibodies by the same low DNA dose were not neutralizing and the epitope pattern was extremely narrow, since it was limited to only one epitope. Thus, EP-based delivery was able to improve the dose efficiency of DNA vaccine and to maintain a highly neutralizing, multi-specific B-cell response, suggesting that it may be an effective approach for chronic hepatitis B therapy at clinically feasible DNA dose.

The development of persistent duck hepatitis B virus infection can be prevented using antiviral therapy combined with DNA or recombinant fowlpoxvirus vaccines.

We recently reported the development of a successful post-exposure combination antiviral and "prime-boost" vaccination strategy using the duck hepatitis B virus (DHBV) model of human hepatitis B virus infection. The current study aimed to simplify the vaccination strategy and to test the post-exposure efficacy of combination therapy with the Bristol-Myers Squibb antiviral drug, entecavir (ETV) and either a single dose of DHBV DNA vaccines on day 0 post-infection (p.i.) or a single dose of recombinant fowlpoxvirus (rFPV-DHBV) vaccines on day 7 p.i. Whilst untreated control ducks infected with an equal dose of DHBV all developed persistent and wide spread DHBV infection of the liver, ducks treated with ETV combined with either the DHBV DNA vaccines on day 0 p.i. or the rFPV-DHBV vaccines on day 7 p.i. had no detectable DHBV-infected hepatocytes by day 14 p.i. and were protected from the development of persistent DHBV infection.

The woodchuck: a model for therapeutic vaccination against hepadnaviral infection.

Interferon-alpha and nucleoside analogues are available for the treatment of chronic hepatitis B virus (HBV) infection but do not lead to a satisfactory result. New findings about the immunological control of HBV during acute infection suggest the pivotal role of T-cell mediated immune responses. Several preclinical and clinical trials were undertaken to explore the possibility of stimulating specific immune responses in chronically infected animals and patients by vaccination. However, vaccination with commercially available HBV vaccines in patients and immunization in woodchucks with core or surface proteins of woodchuck hepatitis virus (WHV) did not result in effective control of HBV and WHV infection, suggesting that new formulations of therapeutic vaccines are needed. Some new approaches combining antiviral treatments with nucleoside analogues, DNA vaccines and protein vaccines were tested in the woodchuck model. It could be shown that therapeutic vaccinations are able to stimulate specific B- and T-cell responses and to achieve transient suppression of viral replication. These results suggest the great potential of therapeutic vaccination in combination with antivirals to reach an effective and sustained control of HBV infection.

The early host innate immune response to duck hepatitis B virus infection.

The early phase after hepatitis B virus infection could play a crucial role in clearance and/or persistence of the virus, particularly in neonates. This work compared the early phase of duck hepatitis B virus infection in 1-day-old (D1) and 28-day-old (D28) ducks to determine whether differences in viral or host innate immune response can be related to the difference in outcome. In the first phase, almost immediately after inoculation, virus was taken up by components of the reticulo-endothelial systems, particularly liver-specific macrophages, Kupffer cells. Very early after infection, the induction of alpha interferon by infected hepatocytes occurred and was rapidly reinforced by recruitment of effector lymphocytes, which directly or indirectly caused apoptosis, eliminating infected hepatocytes, as was seen in mature birds. In addition, a lack of lymphocytic infiltration of the liver was found in D1 ducks, which supports the suggestion that the innate immune network is less effective in D1 ducks. Taken together, these results suggest that failure of the co-ordinated innate immune response rather than a defect in induced antiviral cell-mediated immunity may be the key factor which makes baby ducks vulnerable to persistence of hepadnavirus infection.

Hepadnaviral infection augments hepatocyte cytotoxicity mediated by both CD95 ligand and perforin pathways.

BACKGROUND/AIM: Recently, we documented that hepatocytes can eliminate contacted cells via the CD95 ligand (CD95L)-CD95 pathway and that they are also equipped in perforin and granzyme B and can eradicate other cells via the granule exocytosis mechanism. The aim of this study was to assess whether hepadnaviral infection modifies hepatocyte-mediated cell killing. METHODS: Primary hepatocytes from woodchucks with progressing or resolved hepadnaviral hepatitis and hepatocyte lines transfected with woodchuck hepatitis virus (WHV) genes were examined for cytotoxic effector activity against cell targets susceptible to CD95L and/or perforin-dependent killing. Hepatocytes from healthy animals served as controls. RESULTS: Actively progressing and resolved hepadnaviral hepatitis is associated with a significantly greater capacity of hepatocytes to kill contacted cells. Both hepatocyte CD95L- and perforin-dependent cytotoxicity were augmented. Hepatocytes transfected with WHV X gene, but not those with complete WHV genome or virus envelope or core gene, transcribed significantly more CD95L and perforin and killed cell targets more efficiently. Exposure to interferon-gamma profoundly enhanced hepatocyte cell killing. CONCLUSIONS: Hepatocyte cytotoxic potential is significantly augmented during and following resolution of active hepadnaviral hepatitis. Hepatocyte cytotoxic activity may contribute to both liver physiological functions and the pathogenesis and progression of liver disease, including viral hepatitis.

Intrahepatic expression of genes affiliated with innate and adaptive immune responses immediately after invasion and during acute infection with woodchuck hepadnavirus.

The importance of effective immune responses in recovery from acute hepadnaviral hepatitis has been demonstrated. However, there is no conclusive delineation of virological and immunological events occurring in the liver immediately after hepadnavirus invasion and during the preacute phase of infection. These very early events might be of primary importance in determining the recovery or progression to chronic hepatitis and the intrinsic hepadnaviral propensity to persist. In this study, applying the woodchuck model of acute hepatitis B, the hepatic kinetics of hepadnavirus replication and activation of genes encoding cytokines, cytotoxicity effectors, and immune cell markers were quantified in sequential liver biopsies collected from 1 h postinoculation onward by sensitive real-time cDNA amplification assays. The results revealed that hepadnavirus replication is established in the liver as early as 1 hour after infection. In 3 to 6 h, significantly augmented intrahepatic transcription of gamma interferon and interleukin-12 were evident, suggesting activation of antigen-presenting cells. In 48 to 72 h, NK and NKT cells were activated and virus replication was transiently but significantly reduced, implying that this early innate response is at least partially successful in limiting virus propagation. Nonetheless, T cells were activated 4 to 5 weeks later when hepatitis became histologically evident. Collectively, our data demonstrate that virus replication is initiated and the innate response activated in the liver soon after exposure to a liver-pathogenic dose of hepadnavirus. Nevertheless, this response is unable to prompt a timely adaptive T-cell response, in contrast to infections caused by other viral pathogens.

Enhancement of neutralizing humoral response of DNA vaccine against duck hepatitis B virus envelope protein by co-delivery of cytokine genes.

We explored in the duck hepatitis B virus (DHBV) model the impact of duck interferon gamma (Du-IFNgamma) or interleukin 2 (Du-IL2) co-delivery on humoral neutralizing response induced by DNA-based vaccine encoding DHBV preS/S large envelope protein. Co-delivery of either Du-IL2 or Du-IFNgamma encoding plasmids considerably increased the magnitude of anti-preS humoral response. Moreover, co-administration of cytokine genes led to a significant (p<0.001) enhancement of neutralizing anti-DHBV antibody response, which was more pronounced for Du-IFNgamma. Our data suggest that co-delivery of cytokine and envelope protein encoding plasmids will be a valuable approach for the development of a potent therapeutic DNA vaccine against chronic hepatitis B.

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.

Antiviral therapy with entecavir combined with post-exposure "prime-boost" vaccination eliminates duck hepatitis B virus-infected hepatocytes and prevents the development of persistent infection.

Short-term antiviral therapy with the nucleoside analogue entecavir (ETV), given at an early stage of duck hepatitis B virus (DHBV) infection, restricts virus spread and leads to clearance of DHBV-infected hepatocytes in approximately 50% of ETV-treated ducks, whereas widespread and persistent DHBV infection develops in 100% of untreated ducks. To increase the treatment response rate, ETV treatment was combined in the current study with a post-exposure "prime-boost" vaccination protocol. Four groups of 14-day-old ducks were inoculated intravenously with a dose of DHBV previously shown to induce persistent DHBV infection. One hour post-infection (p.i.), ducks were primed with DNA vaccines that expressed DHBV core (DHBc) and surface (pre-S/S and S) antigens (Groups A, B) or the DNA vector alone (Groups C, D). ETV (Groups A, C) or water (Groups B, D) was simultaneously administered by gavage and continued for 14 days. Ducks were boosted 7 days p.i. with recombinant fowlpoxvirus (rFPV) strains also expressing DHBc and pre-S/S antigens (Groups A, B) or the FPV-M3 vector (Groups C, D). DHBV-infected hepatocytes were observed in the liver of all ducks at day 4 p.i. with reduced numbers in the ETV-treated ducks. Ducks treated with ETV plus the control vectors showed restricted spread of DHBV infection during ETV treatment, but in 60% of cases, infection became widespread after ETV was stopped. In contrast, at 14 and 67 days p.i., 100% of ducks treated with ETV and "prime-boost" vaccination had no detectable DHBV-infected hepatocytes and had cleared the DHBV infection. These findings suggest that ETV treatment combined with post-exposure "prime-boost" vaccination induced immune responses that eliminated DHBV-infected hepatocytes and prevented the development of persistent DHBV infection.

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.

The effect of surgical immunomodulation on liver inflammation and clearance of DHBV infection.

The key to developing a therapeutic vaccine for chronic hepadnavirus infection lies in the characteristics of the host-immune response which leads to clearance of acute infection. Groups of 28-day-old ducks which had been surgically bursectomized (n = 10) or thymectomized (n = 13) on the day of hatch or were untreated (n = 21) were inoculated with 10(9) viral genome equivalents (vge) DHBV, then bled twice a week, and euthanased 40 days later. Serum and liver were tested for DHBV DNA and total leukocytes and peripheral blood mononuclear cells (PBMCs) counted. Liver and spleen sections were either stained with hematoxylin and eosin, and graded for inflammation or stained with peroxidase-labeled anti-human CD3 antibody and examined for T lymphocyte distribution. PBMC counts were similar in all groups. DHBV infection combined with bursectomy increased significantly, while thymectomy decreased significantly the total leukocyte count. The spleen and liver bursectomy increased T lymphocyte number while B cells were decreased. Converse changes were observed in thymectomized ducks. Histological evidence of hepatitis was present in infected control and bursectomized ducks but not in the uninfected control or infected thymectomized ducks. In control animals, DHBV challenge caused viremia in 17 and persistent infection in 11 (56%). Fewer thymectomized ducks (3/13, 23%) and significantly more (100%) bursectomized ducks remained persistently infected (P < 0.001). Unexpectedly, bursectomy led to persistence of infection while clearance of infection occurred normally in thymectomized ducks despite decreased T lymphocyte numbers. This suggests that clearance requires T and B lymphocyte collaboration.

Identification of T-cell epitopes associated with immunity within the surface protein of duck hepatitis B virus.

Duck hepatitis is a convenient model of hepatitis B virus (HBV) infection, but the lack of immunological reagents hampers investigation of pathogenesis and vaccine development. The aim of this study was to define T-cell epitopes in the surface peptide recognized by vaccinated immune birds. Blastogenesis assays were used to test the proliferative response of spleen mononuclear cells to synthetic peptides spanning the pre-S/S region in 22 naïve and 13 immunized and challenged immune ducks. Roughly > or = 50% of the immune ducks responded to five immunodominant peptides eliciting a statistically greater proliferative response than in naïve birds. Fewer ducks responded to an additional six peptides. No statistically significant difference could be shown for the response to 11 peptides between the immune ducks and the naïve ducks. There was no clustering of the immunodominant peptides which were located throughout the surface antigen at sites of major swings in hydrophobicity. A number of peptides which induce lymphoblastogenesis in vaccinated immune ducks have been identified. Their role in spontaneous recovery from duck hepatitis B infection merits investigation.

Vaccination of ducks with a whole-cell vaccine expressing duck hepatitis B virus core antigen elicits antiviral immune responses that enable rapid resolution of de novo infection.

As a first step in developing immuno-therapeutic vaccines for patients with chronic hepatitis B virus infection, we examined the ability of a whole-cell vaccine, expressing the duck hepatitis B virus (DHBV) core antigen (DHBcAg), to target infected cells leading to the resolution of de novo DHBV infections. Three separate experiments were performed. In each experiment, ducks were vaccinated at 7 and 14 days of age with primary duck embryonic fibroblasts (PDEF) that had been transfected 48 h earlier with plasmid DNA expressing DHBcAg with and without the addition of anti-DHBcAg (anti-DHBc) antibodies. Control ducks were injected with either 0.7% NaCl or non-transfected PDEF. The ducks were then challenged at 18 days of age by intravenous inoculation with DHBV (5 x 10(8) viral genome equivalents). Liver biopsies obtained on day 4 post-challenge demonstrated that vaccination did not prevent infection of the liver as similar numbers of infected hepatocytes were detected in all vaccinated and control ducks. However, analysis of liver tissue obtained 9 or more days post-challenge revealed that 9 out of 11 of the PDEF-DHBcAg vaccinated ducks and 8 out of 11 ducks vaccinated with PDEF-DHBcAg plus anti-DHBc antibodies had rapidly resolved the DHBV infection with clearance of infected cells. In contrast, 10 out of 11 of the control unvaccinated ducks developed chronic DHBV infection. In conclusion, vaccination of ducks with a whole-cell PDEF vaccine expressing DHBcAg elicited immune responses that induced a rapid resolution of DHBV infection. The results establish that chronic infection can be prevented via the vaccine-mediated induction of a core-antigen-specific immune response.

Comparative antigenicity and immunogenicity of hepadnavirus core proteins.

The hepatitis B virus core protein (HBcAg) is a uniquely immunogenic particulate antigen and as such has been used as a vaccine carrier platform. The use of other hepadnavirus core proteins as vaccine carriers has not been explored. To determine whether the rodent hepadnavirus core proteins derived from the woodchuck (WHcAg), ground squirrel (GScAg), and arctic squirrel (AScAg) viruses possess immunogen characteristics similar to those of HBcAg, comparative antigenicity and immunogenicity studies were performed. The results indicate that (i) the rodent core proteins are equal in immunogenicity to or more immunogenic than HBcAg at the B-cell and T-cell levels; (ii) major histocompatibility complex (MHC) genes influence the immune response to the rodent core proteins (however, nonresponder haplotypes were not identified); (iii) WHcAg can behave as a T-cell-independent antigen in athymic mice; (iv) the rodent core proteins are not significantly cross-reactive with the HBcAg at the antibody level (however, the nonparticulate "eAgs" do appear to be cross-reactive); (v) the rodent core proteins are only partially cross-reactive with HBcAg at the CD4+ T-cell level, depending on MHC haplotype; and (vi) the rodent core proteins are competent to function as vaccine carrier platforms for heterologous, B-cell epitopes. These results have implications for the selection of an optimal hepadnavirus core protein for vaccine design, especially in view of the "preexisting" immunity problem that is inherent in the use of HBcAg for human vaccine development.

IFN-gamma increases efficiency of DNA vaccine in protecting ducks against infection.

AIM: To detect the effects of DNA vaccines in combination with duck IFN-gamma gene on the protection of ducks against duck hepatitis B virus (DHBV) infection. METHODS: DuIFN-gamma cDNA was cloned and expressed in COS-7 cells, and the antiviral activity of DuIFN-gamma was detected and neutralized by specific antibodies. Ducks were vaccinated with DHBpreS/S DNA alone or co-immunized with plasmid expressing DuIFN-gamma. DuIFN-gamma mRNA in peripheral blood mononuclear cells (PBMCs) from immunized ducks was detected by semi-quantitative competitive RT-PCR. Anti-DHBpreS was titrated by enzyme-linked immunosorbent assay (ELISA). DHBV DNA in sera and liver was detected by Southern blot hybridization, after ducks were challenged with high doses of DHBV. RESULTS: DuIFN-gamma expressed by COS-7 was able to protect duck fibroblasts against vesicular stomatitis virus (VSV) infection in a dose-dependent fashion, and anti-DuIFN-gamma antibodies neutralized the antiviral effects. DuIFN-gamma in the supernatant also inhibited the release of DHBV DNA from LMH-D2 cells. When ducks were co-immunized with DNA vaccine expressing DHBpreS/S and DuIFN-gamma gene as an adjuvant, the level of DuIFN-gamma mRNA in PBMCs was higher than that in ducks vaccinated with DHBpreS/S DNA alone. However, the titer of anti-DHBpreS elicited by DHBpreS/S DNA alone was higher than that co-immunized with DuIFN-gamma gene and DHBpreS/S DNA. After being challenged with DHBV at high doses, the load of DHBV in sera dropped faster, and the amount of total DNA and cccDNA in the liver decreased more significantly in the group of ducks co-immunized with DuIFN-gamma gene and DHBpreS/S DNA than in other groups. CONCLUSION: DHBV preS/S DNA vaccine can protect ducks against DHBV infection, DuIFN-gamma gene as an immune adjuvant enhances its efficacy.

Single B or T-cell epitope-based DNA vaccine using modified vector induces specific immune response against hepadnavirus.

Epitope-based DNA vaccine is an effective and powerful approach against a variety of pathogens or tumors. In present study, we reconstructed a vector that could effectively express short B and T-cell epitope of duck/hepatitis B virus, and investigated the role of the epitope-based DNA vaccination. The pUC19 was modified by inserting the compact transient framework (CTF), including HCMV IE1 promoter, enhancer, Kozak sequence, dual stop codon and 3' terminal bovine growth hormone terminal signal and so on. This modified vector was designated pEC(K) and supposed to effectively express short peptide. A well-defined single B-cell and T-cell epitope encoding gene of duck/hepatitis B virus has been synthesized as candidate epitope and cloned into pEC(K) plasmid, respectively. Transfection of the recombinant DNA into C(2)C(12) cell showed that modified plasmid could effectively express both the single B-cell and T-cell short epitope in the culture supernatant as confirmed by dot immunoblot assay (DIA). The recombinant single B and T-cell epitope-based DNA vaccine was administrated to C57BL/6 mice and could greatly induce specific humoral and CTL response. In addition, the specific antibody against B epitope could specifically bind to the DHBV particles. This report demonstrated that single epitope-based DNA vaccine using modified plasmid vector pEC(K) could induce effective specific immune responses and could be of great use for DNA vaccines.