Study On HIV DNA Vaccine Optimization And DNA Vaccine Technologies

DNA vaccine has become one of the most promising vaccine because of its excellent safety and versatile potential in induction of both humoral and cellular immune responses. However, most present DNA vaccines in clinical trial are deficient mainly because of their relativity low level of immunogenicity. Therefore, how to increase the delivery efficiency of DNA vaccine is a burning question in DNA vaccine study. In this subject we studied on the optimization of DNA vaccine and DNA vaccine delivery technologies.In this project, the unnecessary sequences of our present DNA vaccine vector pDRVISV1.0 were eliminated and new vaccine vector pDRVI4.0 was constructed. Based on pDRVI4.0, DNA vaccine p4.0Env,p4.0Gag,p4.0Pol and p4.0TNR containing HTV-1 Env,Gag,Pol and TNR gene were constructed. Mouse model was introduced to evaluate the immune efficiency between p4.0Env and p1.0Env. The ELISPOT result shows that the deletion of surplus sequences of our present DNA vaccine does not affect its immunogenicityThen bi-cistronic DNA plasmids p4.0AEnvTNR and p4.0AgagPol were constructed on the basis of simplified single-cistronic DNA vaccine. The immunogenicity of each bi-cistronic DNA vaccine and the combination of the two bi-cistronic DNA vaccine with the combination of corresponding single-cistronic plasmids respectively in mouse model. No significant difference was observed between these groups. Besides, influences of different promoter direction on DNA vaccine immunogenicity was studied. Bi-cistronic DNA vaccine plasmids p4.0SEnvPol and p4.0SGagTNR, both of which contain promoters in the same direction,and plasmids p4.0AEnvPol and p4.0AGagTNR, both of which contain promoters in the opposite direction, were constructed. Study in mouse model demonstrates that different promoter direction could not influence the immunogenicity of bi-cistronic DNA vaccine. The bi-cistronic design simplified the present DNA vaccine strategy, reduced the cost and time of production. In the current project.the optimal dosage and of inocculation numbers of gene gun were determined by ELISA assay.The result shows that the optimal dosage of gene gun is 2.25μg and the optimal times is 3 times.Moreover,two intensively discussed DNA delivery methods were studied. BABL/c mice were immunized with p1.0Env through intramuscular injection, gene gun and electroporation routes at different DNA dosages. A luciferase-expressing plasmid was constructed as a reporter and in vivo bioluminescent in BALB/c mice was measured using the IVIS(?) Lumina imaging system.BALB/c mice were vaccinated on 3 inocculations using different delivery methods. The result shows that luciferase expressed in mice delivered 1μg p1.0LUC using gene gun methods was 10 times stronger than that in mice delivered 50μg by using intramuscular injection. HIV-1 Env-specific IgG, IgG1 and IgG2a humoral immnue responses were measured by ELISA and HIV-1 Env-specific cellular immune responses were determined by IFN-y ELISPOT. According to our study, electroporation is an optimal delivery method for our HIV DNA vaccine.