Duck hepatitis B virus (DHBV) shares many basic characteristics with hepatitis B virus (HBV) and is an attractive model for vaccine development.  In this study, DHBV DNA vaccines were designed to express envelope and capsid fusion proteins to enhance the breadth of immune response in ducks.  Attenuated Salmonella typhimurium (SL7207) was used as a carrier and adjuvant to boost the magnitude of immune response.  Based on this strategy, novel DNA vaccines (SL7207-pVAX1-LC and SL7207-pVAX1-SC) were generated.  Growth kinetics, genetic stabilities and relative transcription levels of the L, S and C genes introduced by these vaccine strains were measured before inoculation to guarantee safety and efficacy.  The relative transcript levels of the CD4 and CD8 T genes and the antibody levels (IgY) in ducks receiving the vaccines were higher than those in single gene delivered groups.  Additionally, the copy number of covalently closed circular DNA in hepatocytes after DHBV challenge also provided evidence that our fusion vaccines could enhance the protective efficiency against DHBV infection in ducks.

    The capsid (Cap) protein, which is the only structural protein of duck circovirus (DuCV), is the most important antigen for the development of vaccines against DuCV and the virus’s serological diagnostic methods. In order to use yeast expression system to produce a large quantities of DuCV Cap protein which is close to its natural form to display the antigen peptides perfectly, the Cap gene was optimized into the codon-optimized capsid (Opt-Cap) gene towards the preference of yeast firstly. Then, the genes of Cap and Opt-Cap were separately cloned into pPIC9K plasmid and transformed into Picha pastoris GS115. The strains that displayed the phenotype of Mut+ and contained multiple inserts of expression cassette were selected from those colonies. After the induction expression, the secretory type of Cap protein, which was about 43 kDa, was best expressed under 0.5% (v/v) methanol and sorbitol induction. Compared with the Cap gene, the expression level of Opt-Cap gene was much higher. What’s more, the purified Cap protein had a good reactivity to its specific polyclone antibody and DuCV-positive serum, and it was able to self-assemble into virus-like particles (VLPs). These VLPs, with a diameter of 15–20 nm and without a nucleic acid structure, showed a high level of similarity to DuCV particles in size and shape. All of the results demonstrated that, based on the codon-optimization, it is suitable to use the P. pastoris expression system to produce DuCV VLPs on a large scale. It is the first time that a large amounts of DuCV VLPs were produced successfully in P. pastoris, which might be particularly useful for the further studies of serological diagnosis and vaccines of DuCV.