Comparison of immune response generated against Japanese encephalitis virus envelope protein expressed by DNA vaccines under macrophage associated versus ubiquitous expression promoters.

BACKGROUND: Japanese encephalitis virus (JEV) is the leading cause of viral encephalitis, with ~50,000 cases reported annually worldwide. Vaccination is the only measure for prevention. Recombinant vaccines are an efficient and safe alternative for formalin inactivated or live attenuated vaccines. Nowadays, incorporation of molecular adjuvants has been the main strategy for melioration of vaccines. Our attempt of immunomodulation is based on targeting antigen presenting cells (APC) "majorly macrophages" by using macrosialin promoter. We have compared the immune response of the constructed plasmids expressing JEV envelope (E) protein under the control of aforesaid promoter and cytomegalovirus (CMV) immediate early promoter in mouse model. Protection of immunized mice from lethal challenge with JEV was also studied. RESULTS: The E protein was successfully expressed in the macrophage cell line and was detected using immunofluorescence assay (IFA) and Western blotting. APC expressing promoter showed comparable expression to CMV promoter. Immunization of mice with either of the plasmids exhibited induction of variable JEV neutralizing antibody titres and provided protection from challenge with a lethal dose of JEV. Immune splenocytes showed proliferative response after stimulation with the JEV antigen (Ag), however, it was higher for CMV promoter. The magnitude of immunity provided by APC dominant promoter was non-significantly lower in comparison to CMV promoter. More importantly, immune response directed by APC promoter was skewed towards Th1 type in comparison to CMV promoter, this was evaluated by cytokine secretion profile of immune splenocytes stimulated with JEV Ag. CONCLUSIONS: Thus, our APC-expressing DNA vaccination approach induces comparable immunity in comparison to ubiquitous promoter construct. The predominant Th1 type immune responses provide opportunities to further test its potency suitable for response in antiviral or anticancer vaccines.

Comparative analysis of macrophage associated vectors for use in genetic vaccine.

BACKGROUND: Antigen presentation by non professional antigen presenting cells (APC) can lead to anergy. In genetic vaccines, targeting the macrophages and APC for efficient antigen presentation might lead to balanced immune response. One such approach is to incorporate APC specific promoter in the vector to be used. METHODS: Three promoters known to be active in macrophage were selected and cloned in mammalian expressing vector (pAcGFP1-N1) to reconstruct (pAcGFP-MS), (pAcGFP-EMR) and (pAcGFP-B5I) with macrosialin, EmrI and Beta-5 Integrin promoters respectively. As a positive control (pAcGFP-CMV) was used with CMV promoter and promoterless vector (pAcGFP-NIX) which served as a negative control. GFP gene was used as readout under the control of each of the promoter. The expression of GFP was analyzed on macrophage and non-macrophage cell lines using Flow cytometry and qRT-PCR with TaqMan probe chemistries. RESULTS: All the promoters in question were dominant to macrophage lineage cell lines as observed by fluorescence, Western blot and quantitative RT-PCR. The activity of macrosialin was significantly higher than other macrophage promoters. CMV promoter showed 1.83 times higher activity in macrophage cell lines. The expression of GFP driven by macrosialin promoter after 24 hours was 4.40 times higher in macrophage derived cell lines in comparison with non macrophage cell lines. CONCLUSIONS: Based on this study, macrosialin promoter can be utilized for targeting macrophage dominant expression. In vivo study needs to be carried out for its utility as a vaccine candidate.