Plasmid DNA launches live-attenuated Japanese encephalitis virus and elicits virus-neutralizing antibodies in BALB/c mice

Abstract

We describe novel plasmid DNA that encodes the full-length Japanese encephalitis virus (JEV) genomic cDNA and launches live-attenuated JEV vaccine in vitro and in vivo. The synthetic cDNA based on the sequence of JEV SA14-14-2 live-attenuated virus was placed under transcriptional control of the cytomegalovirus major immediate-early promoter. The stability and yields of the plasmid in E. coli were optimized by inserting three synthetic introns that disrupted JEV cDNA in the structural and nonstructural genes. Transfection of Vero cells with the resulting plasmid resulted in the replication of JEV vaccine virus with intron sequences removed from viral RNA. Furthermore, a single-dose vaccination of BALB/c mice with 0.5 - 5μg of plasmid resulted in successful seroconversion and elicitation of JEV virus-neutralizing serum antibodies. The results demonstrate the possibility of using DNA vaccination to launch live-attenuated JEV vaccine and support further development of DNA-launched live-attenuated vaccine for prevention of JEV infections.

Keywords: DNA vaccine; JE; JEV; Japanese encephalitis virus; Live-attenuated virus; iDNA.

Fig. 1

Preparation of pMG8009 JEV iDNA® vaccine containing synthetic cDNA of SA-14-14-2 JEV strain. (a) Diagram of iDNA construct. Locations of CMV promoter, JEV cDNA, genes, ribozyme and introns 1–3 (filled triangles) are shown not to scale. (b) pMG8009 plasmid stability study in E. coli, by 1% agarose gel electrophoresis. DNA was isolated from eight independent E. coli colonies after passages P1 (lanes 1–8), P5 (lanes 9–16), and P10 (lanes 17–24). To obtain P1 plasmids, pMG8009 was transformed into E. coli Stbl3 cells and grown on LB agar plate containing 50 μg/ml kanamycin. Eight independent colonies from the plate were grown in 2 ml LB cultures and DNA was isolated in 50 μl of sterile water. 1 μl of plasmid was loaded on 1% TAE agarose gel. Then, DNA preparation #1 was transformed into E. coli again and DNA isolations and transformations were repeated for ten times (P2–P10) as described above. M, molecular weight ladder (Thermo). (c) Confirmation of intron removal from viral RNA of pMG8009-derived JEV. PCR was done using pMG8009 as template (lanes 1, 4, 7), viral RNA (lanes 2, 5, 8), and cDNA (lanes 3, 6, 9). PCR was conducted using primers in the vicinity of introns 1 (lanes 1–3), 2 (lanes 4–6) and 3 (lanes 4–8); M, molecular weight ladder.

Fig. 2

Expression of JEV virus in Vero cells transfected with pMG8009 iDNA plasmid. (a) JEV plaques by the infectious center assay (ICA) in Vero cells transfected by electroporation with 1 μg of pMG8009 iDNA plasmid. Representative ICA image shows ~26 ICs and corresponds to approximately 780 IC/μg of iDNA. (b) Western blot using mouse anti-JEV (ATCC VR-1259AF) antibody. Lane M shows SeeBlue Plus 2 protein molecular weight marker (Thermo). Lane 1 shows JEV antigens in pMG8009-transfected Vero cell. Lane 2, untransfected Vero cells. Location of predicted prM, E and NS1 translation products are indicated. (c) Indirect IFA using anti-JEV mouse ATCC VR-1259AF and fluorescein labeled anti-mouse IgG. Panels 1 and 2 show pMG8009-transfected Vero cells and untransfected Vero cells at 160x magnification), respectively. Panel 3 shows pMG8009-transfected Vero cells at 400x magnification. Nuclei of Vero cells are stained in red using propidium iodide counterstain.

Fig. 3

Detection of JEV virus in the medium from Vero cells transfected with pMG8009 iDNA plasmid. (a) Plaque assay in BHK cells. Upper panel, plaque assay of growth medium from virus-infected Vero cells (no electroporation), sample taken on day 7 post-infection with 1000 PFU (sample #6 on Fig. 3b). Lower panel, plaque assay of growth medium from pMG8009-transfected Vero cells (after electroporation), sample taken on day 7 post-transfection with 10 ng of DNA (sample #2 on Fig. 3b). (b) Growth curves of JEV virus in the medium of Vero cells transfected with pMG8009 iDNA (samples 2, 3, 4) or infected with pMG8009-derived virus (samples 5 and 6). Samples 5 and 6 show infection with 10³ PFU of pMG8009-derived vaccine virus of electroporated and non-electroporated Vero cells, respectively.

Fig. 4

Immunogenicity of pMG8009 JEV vaccine in BALB/c mice, by IFA. Mice were vaccinated on day 0 with 5 μg pf pMG8009 plasmid intramuscularly using electroporation. To detect antibodies mice were bled on day 21. Serum from vaccinated mice was used to probe JEV-infected Vero cells in IFA in chamber slides at 1:10 dilution. After incubation with mouse sera, Vero cells were treated with fluorescein-labeled antibodies to mouse IgG (H+L) to visualize cells expressing JEV antigens in green. Slides were covered with mounting medium containing propidium iodide nuclear counterstain and observed under microscope using 160x magnification. Nuclei of Vero cells are stained in red.