The Optimization Of HIV-1 DNA Vaccine And Immunization Approaches

Vaccine is the most efficient and economical measure for controlling and preventing infectious diseases. DNA vaccine as one of the most potential new vaccine strategies, could not only induce cellular and humoral responses efficiently, but also induce long-time immune memory. However, the modest immunogenicity has been considered as the bottle-neck of DNA vaccine industrialization at all times. A series of measures, such as vector optimization, gene modification and in vivo electroporation, were employed in this study, which significantly enhanced the immune responses elicited by HIV-1 DNA vaccine.The experience of our group shows that the manufacture process would become challenging and the efficiency of cell entry would be reduced when the size of the vaccine plasmid exceeds 9kb. To construct multivalent vaccine, starting from the DNA vaccine vector pDRVI1.0, we successfully constructed pDRVI4.0 plasmid vector by removing about 2kb of nonessential sequence from the plasmid backbone and then constructed HIV DNA vaccine based on this new vector. The immunogenicity data in mice showed that compared with pDRVI1.0-based DNA vaccine, the vaccine based on pDRVI4.0 induced the same level of cellular and humoral immune responses. Four codon-optimized antigen gene Env, Pol, Gag and TNR (encode Tat-Nef-Rev fusion protein) which covers 6 viral genes from CN54- a prevalent HIV-1 strain circulating in China, were subcloned into pDRVI4.0 to obtain 4 monocistronic DNA vaccine plasmids p4.0-Env, p4.0-Pol, p4.0-Gag and p4.0-TNR. To further reduce the cost of vaccine production and application, we constructed 2 bicistronic DNA vaccine plasmids p4.0-Env/Pol and p4.0-Gag/TNR based on pDRVI4.0 vector, which carry the same immunogen genes as those in the monocistronic plasmids. Balb/C mice immunized with bicistronic and monocistronic vaccine plasmids in parallel, the results showed that the immune responses induced by bicistronic DNA vaccine have no significant difference with those induced by mixtures of monocistronic DNA vaccine constructs.To further enhance the immune responses induced by the bicistronic DNA vaccine, we modified the antigen genes Pol, Gag and TNR by adding a membrane anchor to the C-termini of them. The new versions of antigens are predicted to be located at the cell surface as membrane-anchored proteins. Moreover, we constructed bicistronic DNA vaccine plasmids p4.0-Env/Pol-TM and p4.0-Gag/TNR-TM which contain these genes. Mice experiments showed that, generally, DNA vaccine-generated antigen-specific humoral and celluar responses could be significantly enhanced by gene modifications relocalizing antigens to the cell surface.To explore whether the direction of expression cassettes on the bicistronic plasmids has any effects on the immune responses, we constructed two bicistronic DNA vaccine plasmids p4.0-Env/Pol-TM-trans and p4.0-Gag/TNR-TM-trans with two expressional cassette placed in opposite directions. Mice experiments showed that, the direction of expressional cassette on bicistronic contructs influence neither the humoral nor the cellular immune responses induced by DNA vaccination.Next, in vivo electroporation delivery was employed to enhance the cell entry efficiency of DNA vaccine plasmids. Luciferase-expressing plasmid p1.0-LUC was administered intramuscularly to Balb/C mice through injection with or without eletroporation. DNA vaccine plasmid p1.0-Env carrying HIV-1 CN54 Env gene was administered to mice through the two approaches mentioned above. Luciferase expression level in murine muscle was detected by IVIS imaging system 24hr after injection. Env-specific antibody immune responses were determined by ELISA. Env-specific celluar immune responses were determined by IFN-γELISPOT. It was shown that luciferase expression level in murine muscle was significantly increased as much as 35 folds through in vivo eletroporation. The immune responses induced by electro-delivered p1.0-Env at 8μg dosage were better than those induced by simple intramusclar injection with 40μg of plasmid DNA. On the other hand, 2 injections followed by electroporation elicited comparable level of humoral and cellular immune responses with those induced by 3 injections without electroporation.In summary, bicistronic DNA vaccine could reduce number and size of DNA vaccine constructs substantially without compromising their immunogenicity. Humoral and cellular responses induced by DNA vaccination could be enhanced significantly by gene modifications to relocalize the antigens to cell surface as transmenbrane proteins. As a new gene delivered approach, in vivo electroporation could enhance both the gene expression and the immune responses mediated by plasmid DNA in mice.