Codon Optimization And Molecule Adjuvant Enhances Immune Responses Of Newcastle Disease Virus F-DNA Vaccine

Newcastle disease (ND) is a highly contagious viral disease of poultry and other bird species caused by Newcastle disease virus (NDV), its effects are most notable in domestic poultry due to their high susceptibility and the potential for severe impacts of an epidemic on the poultry industries. the conventional vaccine have played an important role to prevent and to control ND in practice,and the most widely used was NDV live vaccine, but both attenuated vaccine and inactivated vaccine can result in latent infection and exist serious defects that can not distinguish vaccine immunization and natural infection . so developing ND engineering vaccine is of great importance to prevent this disease.DNA vaccine which has various advantages is considered as one of the most potential engineering vaccine . this study two approaches were used to enhance the immunogenicity:codon-optimization of NDV F48E9 strain F gene inserts and C3d conjugation to F or combined immunization with IL-2 in order to select low dosage, economic and have commercial potential NDV DNA vaccine.First, we optimized the F gene of NDV F48E9 strain according to the chicken bias codon usages and adding Kozak sequence at the upstream . the optimized F (optiF) and the wild-type F genes were inserted into the pVAX1vector, respectively. The F protein transient expressed in COS 7 cells could be detectable by indirect immunofluorescence and Western-blot at 72 h pos-transfection. The results showed that the expression levels of optiF in vitro was considerably increased in comparison to that of the respective wide-type sequence in the same vector. Two-week-old SPF chickens were inoculated by multi-point injection of plasmids pVAX1, pVAX1-F and pVAX1-opti F, respectively, 200μg for each chichen, and the control group was injected with PBS alone. followed by booster immunization with the same dose at the two week after primary immunization. Two weeks later, the chickens were challenged by NDV F48E9 velogen strain with a dose of 105EID50, then calculating their morbidity and mortality. Blood samples were collected weekly for detecting the antibody directly against the F protein; peripheral blood lymphocytes were isolated and probed for the composition of CD4+,CD8+ and TCRγδT cells, and splenic lymphocytes proliferation assay for measuring cell-mediated immunity was done. The chickens vaccinated by pVAX1-optiF were found to be earlier F protein antibody production, significantly enhanced stimulation index and marked increase in percentage of CD4+,CD8+ and TCRγδT cells, comparing to that vaccinated by pVAX1-F. And the chickens of optiF vaccine group were completely protected from virus challenge. The results indicated that codon optimization of F gene could observably enhance the protection efficiency of NDV F48E9 strain DNA vaccine.we further investigate the immunopotentiation effects of molecular adjuvant C3d and IL-2 to NDV F-DNA vaccine at low dosage.so this study we construct various expression vectors: pV-optiF expressing F protein alone, pV-C3dn-optiF(n=1-6)fused to one to six copies of chicken C3d gene and pV-IL-2 expressing chicken IL-2. pV-optiF,pV-C3d-optiF and pV-C3d5-optiF were selected for this research based on the result of in vitro transient expression and pre-exprement .chicken were inoculated with decreasing doses (200μg, 50μg or 20μg)of these plamids respectively,another three groups inoculated with decreasing doses (200μg, 50μg or 20μg)of pV-optiF and co-immunization with 100μg plamid pV-IL-2 at the same time. Chicken vaccinated with the highest dose (200μg) of DNA had high anti-F protein specific antibody titers regardless of the addition of C3d and IL-2. At lower doses (50μg and 20μg) of DNA, chicken vaccinated with optiF fused C3d or co-immunization with IL-2 had enhanced immune responses compared to chicken vaccinated with DNA expressing F only. The protective humoral immunity, cellular immunity and immune protective effect against virulent virus of chicken vaccinated with pV-C3d-optiF or pV-optiF co-immunization with IL-2 are better than other plamids.In addition, chicken vaccinated with pVAX1-C3d-optiF at 20μg of DNA or vaccinated with pVAX1-optiF at 200μg and co-immunization with 100μg pVAX1- IL-2 obtain the optimum efficiencyIn conclusion,codon optimization and fusion one copy of C3d or co-immunization with plamid which express IL-2 can remarkably improve the immune response of NDV F-DNA vaccine, these approaches could function in a synergistic manner result in protect chickens from lethal NDV virus challenge, even at the low dose of 20μg, implied the potential possibility for the commercialization of ND DNA vaccine in the future. pVAX1-C3d-optiF and pVAX1-optiF+IL-2 have good immune effect and with low costs hoping to become an efficient and safe new ND engineering vaccine.