The Effect Of Codon Optimization On The Immunogenicity Of Middle Hepatitis B Surface Antigen Dna Vaccine

Background: Hepatitis B is the most prevalent infections diseases in our country which caused a grave threat to the public health. HBV DNA vaccine is a new hope for preventing and curing of hepatitis B due to the fact that it can elicit sustained cell mediated as well as humoral immune responses. However, immunogenicity of the HBV DNA still need to be enhanced.There is a great difference in codon usage between the pathogen and mammals which may be one of the limitations of heterologous protein expression in mammals host cells. Low protein expression has not enough effective stimulation to induce immune responses. This may be a major obstacle in DNA development. Codon usage optimization basically involves altering the rare codons in the target gene so that they more closely reflect the codon usage of the host without modifying the amino acid sequence of the encoded protein. It has been proved that this method is effective in many system increasing protein expression in vitro and antigen specific responses in vaccinated animals, for example, HPV, HIV and Influenza virus DNA vaccine. In the current study, we have developed a codon-optimized DNA vaccine encoding middle hepatitis B surface antigen (MHBs). At the same time, we have attempted to investigate whether codon optimization of the MHBs antigen gene could enhance the expression and the immunogenicity of the MHBs DNA vaccine. This is very important for developing a new HBV DNA vaccine with more immunogenicity.Objective: To investigate whether codon optimization of the middle hepatitis B surface antigen gene could enhance the expression in vitro and immunogenicity of the MHBs DNA vaccine.Methods: According to the sequence of amino acids of MHBs(adr subtype), the codon optimized genes were designed and synthesized without changing the sequence of amino acids and then cloned into vector pSW3891. It was named pSW3891/MHBs/adr/opt. 293T cells were transiently transfected with opt and wild-type MHBs DNA vaccine (pSW3891/MHBs/adr) and empty vector pSW3891. The protein expression level was assessed by western blot. Mice used in the studies were from 6 to 8 weeks of age. Each of the BALB/c mice was intramuscularly injected , at 0, 2, 4 and 6 weeks, in the right and left tibialis anterior muscles, with 100μg of plasmid opt DNA or adr DNA. The negative control group was injected with the same volume of an empty pSW3891 vector. Sera from immunized mice were collected by orbital bleeding before the first immunization and 2 weeks after every immunization. Anti-HBs in sera was tested by ELISA. Immunized mice were sacrificed 2 weeks after the last immunization, and spleens were removed aseptically. IFN-γsecretion splenocytes of mice immunized with opt, adr and pSW3891 were tested by ELISPOT.Results: It was proved that codon-optimized DNA vaccine encoding the MHBs was constructed correctly by digesting with BamH1 and Pst1 and direct sequencing. The level of protein expression in both supernatant and lysate of 293T cells transfected with opt is higher than with adr. Specific anti-HBs antibody could be detected in the sera from immunized mice with opt and adr at two weeks after the first immunization. The OD values of the anti-HBs antibody in the sera from immunized mice with opt is stronger than with adr at the same test points. The specific antibody reached the peak at week 8 both in groups with opt and adr. The peak antibody titer in the sera from immunized mice with opt (1:364500) is superior to with adr (1:121500) . The BALB/c mice immunized with opt showed more antigenic specificity IFN-γ secretion splenocytes(165.9 /5×105 cells) than adr (90.7 /5×105 cells).Conclusion: Codon optimization of MHBs DNA vaccine could improve protein expression and induce significantly humoral and cellular immune responses in BALB/c mice.