A single intramuscular injection of recombinant plasmid DNA induces protective immunity and prevents Japanese encephalitis in mice

Abstract

Plasmid vectors containing Japanese encephalitis virus (JEV) premembrane (prM) and envelope (E) genes were constructed that expressed prM and E proteins under the control of a cytomegalovirus immediate-early gene promoter. COS-1 cells transformed with this plasmid vector (JE-4B clone) secreted JEV-specific extracellular particles (EPs) into the culture media. Groups of outbred ICR mice were given one or two doses of recombinant plasmid DNA or two doses of the commercial vaccine JEVAX. All mice that received one or two doses of DNA vaccine maintained JEV-specific antibodies 18 months after initial immunization. JEVAX induced 100% seroconversion in 3-week-old mice; however, none of the 3-day-old mice had enzyme-linked immunosorbent assay titers higher than 1:400. Female mice immunized with this DNA vaccine developed plaque reduction neutralization antibody titers of between 1:20 and 1:160 and provided 45 to 100% passive protection to their progeny following intraperitoneal challenge with 5,000 PFU of virulent JEV strain SA14. Seven-week-old adult mice that had received a single dose of JEV DNA vaccine when 3 days of age were completely protected from a 50, 000-PFU JEV intraperitoneal challenge. These results demonstrate that a recombinant plasmid DNA which produced JEV EPs in vitro is an effective vaccine.

FIG. 1

Map of the JEV genomic structure (top) and the DNA sequence of oligonucleotides used in RT-PCR to construct the transcription unit for the expression of prM-E protein coding regions (bottom). Potential transmembrane helices of viral polyprotein are indicated by blackened areas.

FIG. 2

Schematic representations of plasmid vectors pCDNA3, pCBamp, and pCIBamp. These plasmids include the CMV promoter/enhancer element, BGH poly(A) signal and transcription termination sequence [BHGp(A)], ampicillin resistance gene (Amp), and ColE1 ori for selection and maintenance in E. coli. The f1 ori for single-stranded rescue in E. coli cells, SV40 ori, neomycin coding region, and SV40 poly(A) [SV40 p(A)] sequences were deleted from pCDNA3 to generate pCBamp. An intron sequence was inserted in the NcoI-KpnI site of pCBamp to generate pCIBamp. The multiple cloning site for the insertion of JEV genes, located between the TATA box of the CMV promoter/enhancer and BHG poly(A) site, is shown.

FIG. 3

JEV-specific reactivity of prechallenge and postchallenge serum samples obtained from mice immunized with DNA vaccine or JEVAX. Serum specimens collected from the mice used in the experiments represented in Tables 3 and 4 were randomly selected and tested at 1:1,000 dilution by Western blot analysis using purified JEV as the antigen. pCDJE2-7x2-S was the serum from one of the mice challenged at 4 days of age (Table 4). NMAF, 4G2-AF, and JEV HIAF were the mouse ascitic fluids included as normal mouse, E-specific, and JEV hyperimmune controls, respectively.

FIG. 4

Postchallenge survival rates of mice (10 per group) that were immunized with pCDJE2-7, pCBJE1-14, pCIBJES14, pcDNA3/CAT, or JEVAX at 3 days of age and challenged i.p. with 50,000 PFU of JEV (SA14) 7 weeks postimmunization. A P value of 0.003 was obtained by Fisher's exact test when the survival rate of the JEV DNA-immunized groups was compared with that of the pcDNA3/CAT or JEVAX group.

FIG. 5

Graphic representation, generated by the TMHMM program, indicating probable orientations of five transmembrane helices in the prM-E protein expressed by pCDJE2-7 (A), pcDNA3JEME (B), and pJME (C). ER, endoplasmic reticulum.