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

Abstract

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.

Figure 1

Restriction analysis of the constructs. Independent run gel documented in 1% Agarose in TAE buffer. M: 1 Kb+ Ladder (Invitrogen); 1: pCMV-E with SacI digested (Fragments: 4999 & 1812 bp); 2: pMS-E with VspI + NotI digested (Fragments: 2926 + 1551 + 703 bp); 3: pNIX-E (Undigested).

Figure 2

Western Blot Analysis. (A) 10% SDS-PAGE gel (B) Western blot analysis of the total cell lysates of the RAW 264.7 cells. M: PageRuler™ (Fermentas); 1: pCMV-E; 2: pMS-E; 3: pNIX-E. The blot shows expressed prM and E protein from different constructs after 24 hours of transfection.

Figure 3

Detection of E protein using IFA. Expression of E protein in transfected RAW264.7 cells were detected 24 hrs post transfection. Figures (A) pCMV-E and (B) pMS-E confirms the expression of E protein whereas Fig. (C) pNIX-E and (D) Untransfected were used as a negative control.

Figure 4

ELISA. Antibody response of BALB/c mice immunized with plasmid DNA of different constructs by intramuscular injection. First booster dose was given after 21 days of first immunization and second booster after 36 days. Serum samples were collected on the given days and stored at -70. Each column indicates the mean response ± SEM.

Figure 5

Virus neutralization assay. Neutralization assay of sera from mice immunized with different constructs. The highest dilution of mice sera that resulted into more than 50% CPE was considered. As a positive control JEV immune peritoneal fluid was used whereas peritoneal fluid from the non immunized mice was used as a negative control.

Figure 6

Production of cytokines upon immunization of different constructs. CBA was performed with the supernatant for Th1 and Th2 cytokines. The graph represents the concentration of cytokines TNF, IFN-γ, IL-2, IL-4 and IL-5. The data presented here are the means ± SEM of cytokine profile after (A) Second dose, (B) third dose and (C) the ratio of IFN-γ and IL-4 to show the skewness of immune response.

Figure 7

Lymphocytes proliferation assay. Lymphocytes from mice group immunized with different vaccine constructs were incubated with various amount of JEV Ag. After each dose the proliferative response was evaluated. Lymphocytes from unimmunized and pNIX-E were used as negative controls. Proliferation index were calculated from the means of experiments in triplicate.