Immune system has devised modalities to overcome nonspecific, autoreactive immune response while optimally maintaining the balance. Thus, immune response through antigen presentation by non-hematopoitic cells would actually be downregulated, while immune response generated through professional APC would be long term and balanced. Failure to have second signalling, which lack in non professional APC may lead to reduced immune response or even anergy . The promoter of CMV is commonly used in mammalian expression system due to its strong activity in large varieties of cells . The preference of gene expression is based on its application; in gene therapy constitutive expression is favoured . On the contrary, to enhance the immune response of DNA vaccine, gene expression was restricted only to professional APC  and sometimes limited as a safety concern . DC is considered to be the most potent and remained as highly preferred target cell. Although in a study, targeting DC alone was reported insufficient .
After ex vivo evaluation of promoters active in macrophages , we have selected the highest expressing constructs (macrosialin) and extended our study for in vivo analysis of macrosialin along with CMV promoter for comparison. JEV E protein expressed under these promoters was used for evaluation of concept in terms of antigen specific immune response. Induction of virus neutralizing antibodies, protection from lethal challenge and cytokine profile of immune splenocytes was studied and compared. E protein of JEV is considered to be a vital protein to target, because antibody against E protein has shown to neutralize JEV infectivity and also it is functionally important [12, 56].
All the plasmid constructs were tested and confirmed to drive the expression of E gene of JEV before in vivo inoculation. In transfection experiment, Western blot and IFA analysis showed the constructs under study expressed E gene with high efficiency in RAW 264.7 cells. This indicated that the plasmid could be used further for subsequent in vivo experiments. Though we have shown the expression of E protein in RAW264.7 cells as a qualitative test, still the noticeably higher expression is seen with pCMV-E construct. We had shown similar results quantitatively in our earlier study using GFP reporter system .
To explore the applicability of these constructs as vaccine, groups of mice were vaccinated intramuscularly with recombinant plasmids. In addition, to evaluate prime boost models, administration of plasmid followed by MbrAg as a booster was used. All constructs, pCMV-E, pMS-E and pCMV-E/MbrAg (test groups) successfully led to the production of anti-E Ab. As measured by ELISA, the Ab produced in pCMV-E and pMS-E were nearly similar after the third dose, but increased significantly in pCMV-E/MbrAg groups. To measure the neutralizing abilities of these Ab, virus neutralization test was carried out. pCMV-E/MbrAg group showed the highest neutralizing Ab titre in all sera, whereas pCMV-E and pMS-E showed similar titre after the second dose, after the third dose the neutralizing Ab titre increased significantly in pCMV-E groups. The increase in Ab titre after subsequent dose shown by ELISA and neutralizing test was similar in comparison shown by other group (21).
For the evaluation of cellular response, lymphocyte proliferation assay was carried out. Significant proliferative response was observed in the splenocytes of vaccinated mice when compared with either pNIX-E or PBS inoculated mice. Similar proliferation indices were seen in the group of pCMV-E and pMS-E, whereas pCMV-E/MbrAg showed the highest response.
Cytokine has a major role to play in immune response against viruses, through direct antiviral activity as well as directing an array of immune responses to control the infection . We studied the responses after immunization governed by different promoters through cytokine secretion profile of spleen cells. The result indicated that the magnitude of cytokine production was higher in pCMV-E in comparison to pMS-E at all times. The cytokine level for pCMV-E/MbrAg group increased dramatically after the second dose since it was boosted by MbrAg which acted as a better immunogen. This finding is consistent with previous studies which have demonstrated that DNA priming followed by protein boosting results in greater humoral response and mixed Th1/Th2 response with higher IFN-γ, IL-2 and IL-4 cytokines [58, 59]. After the third dose increase in Th1 cytokine might have downregulated the Th2 cytokines. To evaluate the ratio of Th1 and Th2 responses, we observed the ratio of IFN-γ/IL-4, interestingly we observed macrosialin promoter highly skewed the response towards Th1 cytokine in comparison to CMV promoter. Our result for dominant Th1 response after i.m. inoculation of plasmid is in agreement with the earlier observation , moreover it also supports the previously observed Th1 biased response with APC targeted Ag delivery [54, 60, 61].
To assess the efficacy of vaccine in terms of protection, mice were challenged with lethal dose of JEV. We observed 87.5% protection in pCMV-E group and also for pCMV-E/MbrAg group, whereas it was 75% in pMS-E group. For protection to the host against JEV, neutralizing Ab plays a significant role . For viral infections the importance of IFN-γ has been amply demonstrated . Comparatively lower level of protection in pMS-E could be attributed to lower neutralizing Ab titre and lower IFN-γ levels (after second dose) and its non significant rise after the third dose. TNF is a proinflamatory cytokine considered efficient in stimulating DC maturation, migration and induction of proliferative and cytolytic activity of T cells and NK cells , for pMS-E lower TNF level was observed in comparison to pCMV-E or pCMV-E/MbrAg. With the acceptable difference in overall immune response in the constructs, it is considered as evidence of protection if the neutralizing titres are ≥ 1:10 .
In conclusion, CMV and Macrophage active promoter constructs resulted in successful expression of the E protein after intramuscular (i.m.) immunization. The expression level of pMS-E was lower than those obtained with the use of pCMV-E constructs but sufficient to induce protection in mouse model.