Optimization Of DNA Vaccine Vector And Research On New DNA Vaccine Adjuvant

DNA vaccine not only has advantages of live attenuated vaccine,but also has safety of inactivated vaccine,and could induce both humoral and cellular immune responses. Antigen-encoding naked DNA is relatively safe,stable and easy to produce,and can mediate sustained antigen expression in vivo.However,the development of DNA vaccine candidates has been limited due to their relatively modest immunogenicity.To enhance the expression level of antigen is an important method for improving the immunogenicity of DNA vaccine.There are several ways to enhance antigen expression, such as optimization of the expression regulatory elements in the plasmid backbone, optimization of the codon usage of the antigen gene.For most vaccine plasmids,the human CMV immediately early intronA is a common choice as an expression regulatory element because it promotes intronless gene expression at posttranscriptional level when combined with the hCMV IE promoter/enhancer elements.It has been shown that post-transcriptional regulatory elements(PREs) of Hepatitis B virus(HPRE) and Woodchuck Hepatitis virus(WPRE),constructive transport element(CTE) of Mazon-Pifzer monkey virus and pre-mRNA processing enhancer(PPE) of herpes simplex virus' kinase(HSV-TK) gene are cis-acting RNA elements that can increase the accumulation of cytoplasmic mRNA of an intronless gene by promoting mRNA exportation from the nucleus to the cytoplasm,enhancing 3' end processing and stability. Whether these post-transcriptional regulatory elements mentioned above could substitute for hCMVIE intronA or have a synergistic effect with it is unknown.In our study,the plasmid vector pCMV was constructed from pVR1012 through deleting the hCMVIE intronA.Then the HPRE,WPRE,CTE and PPE elements were inserted into pVR1012 or pCMV backbone to generate the vectors pIn-HPRE,pIn-WPRE,pIn-CTE,pIn-PPE and pHPRE,pWPRE,pCTE,pPPE,respectively.To compare of expression enhancing effect of hCMV IE intronA and HPRE,WPRE,CTE,PPE,luciferase gene was subcloned into the vectors mentioned above,then plasmids were transfected into 293T cells.As shown by transient transfection assay,pIn-HPRE and pIn-WPRE vectors could significantly enhance the luciferase gene expression in vitro,although pHPRE and pWPRE vectors had no differences compared with pVR1012.To compare both the gene expression-enhancing and immunogenicity-improving effects of HPRE,WPRE with or without hCMVIE intronA on the DNA vaccine constructs,the wild-type/codon-optimized HIV-1 CN54 Gag coding sequence were used as a model antigen and subcloned into vectors containing HPRE,WPRE,respectively.Then 293T cells were transiently transfected with plasmids and Balb/C mice were immunized with HIV-1 Gag DNA vaccine.The results showed that,HPRE significantly improved Gag gene expression in 293T cell.The highest level of gene expression was observed when both hCMVIE intronA and HPRE were present in the same plasmid.Furthermore,pInHGagw (+intronA/+HPRE) at 10μg dose could induce higher Gag specific immune response level than that induced by pGagw(-intronA/-HPRE) or pInGagw(+intronA/-HPRE) at 40μg dose.Vector optimization had less effect on enhancing codon optimized gene expression both in vitro and in vivo,but our data also demonstrated that wild type gene constructs could also achieve in vitro expression and in vivo immunogenicity which were comparable to that the same amount of codon-optimized Gag DNA vaccine did if the vector constructs were optimized by hCMVIE intronA and HPRE.A large number of researches demonstrated that the use of adjuvant is an important method for improving the immunogenicity of DNA vaccine.Mycobacterium boris bacillus Calmette-Gu(?)rin(BCG) cell-wall skeleton consists of mycolic acids, arabinogalactan and peptidoglycan and activates at least five kinds of TLRs(TLR1, TLR2,TLR4,TLR6 and TLR9),and is rich of CpG motif which is connecting with immunomodulatory effects.So it is a strong non-specific immunostimulator and is effective in some vaccine researches.Polysaccharide nucleic acid fraction from BCG (BCG-PSN) is an immunomodulator extracted by hot phenol method from BCG,and is considered to be successful clinical application in immune disorders,immune levels decline and the treatment of allergic diseases.In the present study,we tried to evaluate the BCG-PSN as adjuvant to augment the cellular and humoral immune responses as well as the generation of long-lasting immune memories by vaccinating together with various doses of pDRVI1.0gp1455M(HIV-1 CN54 gp145 gene) following different immunization schedules.The results showed that,BCG-PSN could significantly improve the immunogenicity of DNA vaccine when co-immunized with DNA vaccine.Further, we demonstrated that the use of BCG-PSN could reduce the times of DNA vaccination and the amount of DNA vaccine.Our study provides a new method for improving the immunogenicity of DNA vaccine, and has established a strong foundation for developing DNA vaccine for Chinese HIV strain.