The Expression And Immunogenicity Of DNA Vaccine Encoding Middle Hepatitis B Surface Antigen With Relocating N-linked Glycosylation

Objective: To investigate the expression and immunogenicity of DNA vaccine encoding middle hepatitis B surface antigen with relocating N-linked glycosylation.Methods: Site-directed mutagenesis were used to construct four DNA vaccines encoding middle hepatitis B surface antigen with relocating N-linked glycosylation named Adr-dN4, Adr-N4-5, Adr-N4-6 and Adr-N4-7, which transfer N-linked glycosylation sites from amino acid 4 to 5, 6 or 7 in preS2 domain. HEK 293T cells were transiently transfected with Adr-dN4, Adr-N4-5, Adr-N4-6, Adr-N4-7, wild-type MHBs DNA vaccine (pSW3891/MHBs/Adr) and pSW3891 vector. The protein was measured by western blot. 6 to 8 weeks age BALB/c mice were used in the experiments. Each BALB/c mouse separately was intramuscularly injected with 100μg of each mutant or Adr in the right and left tibialis anterior muscles at 0, 2, 4 and 6 weeks. The control group were injected with the same dose of vector pSW3891. Sera from immunized mice were collected by orbital bleeding at pre-immunization and 2 weeks after last immunization. Anti-HBs in sera of mice were detected by ELISA. Immunized mice were sacrificed 2 weeks after the last immunization and the spleens were removed aseptically. Peptide of HBsAg specific IFN-γsecreted splenocytes of mice were detected by ELISPOT.Results: It was shown that DNA vaccine encoding middle hepatitis B surface antigen with relocating N-linked glycosylation was successfully constructed and comfirmed by digesting with BamHⅠand PstⅠand direct sequencing. There are MHBs in the lysates of 293T cells transfected with each vaccine by western blot and in the supernatants in Adr, Adr-N4-5, Adr-N4-7. Anti-HBs could be detected in the sera from immunized mice with all vaccines at two weeks after the primary immunization. Anti-HBs reached the peak at week 8 both in all groups. The peak antibody titer in the sera from immunized mice with Adr (1:121500) is slightly superior to with Adr-dN4, Adr-N4-5, Adr-N4-6, Adr-N4-7. But there was no significant difference between Adr and Adr-N4-5, Adr-N4-7 (P>0.05), while there was significant difference between Adr and Adr-dN4, Adr-N4-6 (P<0.05). The BALB/c mice immunized with Adr showed more antigenic specific IFN-γsecreted splenocytes (138.6/5×105 cells) than Adr-dN4 and Adr-N4-6.Conclusion: DNA vaccine encoding middle hepatitis B surface antigen with relocating N-linked glycosylation was successfully constructed. N-linked glycosylation on amino acid 4 in preS2 domain plays a crucial role in secreting of MHBs from eukaryotic cells,and defect by deleting N-linked glycosylation on amino acid 4 can be rescued by adding N-linked glycosylation on amino acid 5 or 7. Secreting of MHBs from the cells is essential to induce the specific cellular and humoral immunity. DNA vaccines encoding middle hepatitis B surface antigen with relocating N-linked glycosylation on amino acid 5 or 7 can induce similar cellular and humoral immune responses as Adr.