Screening of protective antigens of Japanese encephalitis virus by DNA immunization: a comparative study with conventional viral vaccines

Abstract

In this study, we evaluated the relative role of the structural and nonstructural proteins of the Japanese encephalitis virus (JEV) in inducing protective immunities and compared the results with those induced by the inactivated JEV vaccine. Several inbred and outbred mouse strains immunized with a plasmid (pE) encoding the JEV envelope protein elicited a high level of protection against a lethal JEV challenge similar to that achieved by the inactivated vaccine, whereas all the other genes tested, including those encoding the capsid protein and the nonstructural proteins NS1-2A, NS3, and NS5, were ineffective. Moreover, plasmid pE delivered by intramuscular or gene gun injections produced much stronger and longer-lasting JEV envelope-specific antibody responses than immunization of mice with the inactivated JEV vaccine did. Interestingly, intramuscular immunization of plasmid pE generated high-avidity antienvelope antibodies predominated by the immunoglobulin G2a (IgG2a) isotype similar to a sublethal live virus immunization, while gene gun DNA immunization and inactivated JEV vaccination produced antienvelope antibodies of significantly lower avidity accompanied by a higher IgG1-to-IgG2a ratio. Taken together, these results demonstrate that the JEV envelope protein represents the most critical antigen in providing protective immunity.

FIG. 1

(A) Schematic diagram of plasmid constructs encoding various JEV structural and nonstructural proteins. The first and last nucleotide positions of each gene are shown above the JEV genome. (B) Immunoblot analysis. Cellular proteins of transfected cells were subjected to SDS-PAGE followed by blotting onto nitrocellulose. Nitrocellulose strips were reacted with anti-E, anti-NS1, and anti-NS3 MAbs as indicated and detected with HRP-conjugated second-step antibodies. (C) In vitro-coupled transcription-translation reaction. The gene products produced from various plasmid constructs in the presence of [³⁵S]methionine were analyzed by SDS-PAGE and exposed to X-ray film.

FIG. 2

Kinetics of anti-E antibodies in C3H/HeN mice immunized with DNA or inactivated JEV vaccines. C3H/HeN mice were given intramuscular (i.m.) or gene gun injections of plasmid pE or pcDNA3 three times at 3-week intervals. The inactivated JEV vaccine was administered intraperitoneally and boosted once 3 weeks later. Sera were obtained at different time points and assayed for the presence of JEV E-specific antibodies. The concentration of anti-E antibodies was calculated from the standard curve generated from serially diluted control antibodies and expressed as units per milliliter. The data are presented as means ± standard deviations for five animals per time point.

FIG. 3

Mice immunized with DNA or inactivated viral vaccines protected from lethal JEV challenge. Groups of C3H/HeN mice were immunized with the inactivated JEV vaccine or plasmid pE or pcDNA3 by intramuscular (i.m.) or gene gun injections as detailed in the legend to Fig. 2. Two weeks after the last immunization, mice were challenged with 50 LD₅₀s of JEV Beijing-1 as described in Materials and Methods. Following challenge, mice were observed for 30 days and the percentage of survivors was calculated.

FIG. 4

Isotypes of anti-E antibodies generated by DNA or conventional viral vaccines. Groups of C3H/HeN mice were immunized with DNA or inactivated JEV vaccines as described in the legend to Fig. 2 or were sublethally immunized twice at 3-week intervals with 6.0 × 10⁵ PFU of JEV Beijing-1 as described in Materials and Methods. Two weeks after the last immunization, mice were challenged with 50 LD₅₀s of JEV Beijing-1. Sera were collected prechallenge (A) and 2 weeks postchallenge (B) and analyzed for the presence of IgG1 and IgG2a anti-E antibodies. Concentrations of IgG1 and IgG2a anti-E antibodies were presented as end-point titers defined as the highest serum dilution that resulted in an absorbance value two times greater than that of nonimmune serum with a cutoff value of 0.05. Samples below the limit of detection were assigned a value of 10. The titer of individual animals and the mean titers of each immunized group are presented. i.m., intramuscular.

FIG. 5

Avidity of anti-E antibodies generated by DNA or conventional viral vaccines. Mice were immunized as described in the legend to Fig. 4. Sera collected two weeks after the last immunization were analyzed for antibody avidity. The tested sera from each group were adjusted to contain equal anti-E titers before use. The avidity is reported as the reciprocal dilution of inactivated JEV particles added to the well that resulted in a 50% binding inhibition of each immune serum sample (I₅₀). Data represent the mean I₅₀ ± standard deviation for five animals in each group from one representative experiment of three performed. i.m., intramuscular.

FIG. 6

Effect of injection numbers on DNA vaccine-induced protective immunities. C3H/HeN mice were given an intramuscular injection of pE either one, two, or three times at 3-week intervals. Mice immunized intramuscularly with pcDNA3 three times at 3-week intervals served as negative controls. Animals that received one dose of DNA immunization were challenged with 50 LD₅₀s of JEV Beijing-1 at week 3, and those that received two or three DNA inoculations were challenged 2 weeks after the last immunization. Following challenge, mice were observed for 30 days and the percentage of survivors was calculated.

FIG. 7

Anti-E antibody and protective immunity induced by JEV DNA vaccination in different mouse strains. Groups of BALB/c and ICR mice were immunized intramuscularly with plasmid pE or pcDNA3 and challenged with a lethal dose of JEV as detailed in the legend to Fig. 3. The anti-E antibody at different time points (A) and the percentage of survivors (B) in each immunized group are shown.