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. 2003 Mar;77(6):3487-94.
doi: 10.1128/jvi.77.6.3487-3494.2003.

A Japanese encephalitis virus peptide present on Johnson grass mosaic virus-like particles induces virus-neutralizing antibodies and protects mice against lethal challenge

Manisha Saini  1 , Sudhanshu Vrati
Affiliations

A Japanese encephalitis virus peptide present on Johnson grass mosaic virus-like particles induces virus-neutralizing antibodies and protects mice against lethal challenge

Manisha Saini et al. J Virol. 2003 Mar.

Abstract

Protection against Japanese encephalitis virus (JEV) is antibody dependent, and neutralizing antibodies alone are sufficient to impart protection. Thus, we are aiming to develop a peptide-based vaccine against JEV by identifying JEV peptide sequences that could induce virus-neutralizing antibodies. Previously, we have synthesized large amounts of Johnson grass mosaic virus (JGMV) coat protein (CP) in Escherichia coli and have shown that it autoassembled to form virus-like particles (VLPs). The envelope (E) protein of JEV contains the virus-neutralization epitopes. Four peptides from different locations within JEV E protein were chosen, and these were fused to JGMV CP by recombinant DNA methods. The fusion protein autoassembled to form VLPs that could be purified by sucrose gradient centrifugation. Immunization of mice with the recombinant VLPs containing JEV peptide sequences induced anti-peptide and anti-JEV antibodies. A 27-amino-acid peptide containing amino acids 373 to 399 from JEV E protein, present on JGMV VLPs, induced virus-neutralizing antibodies. Importantly, these antibodies were obtained without the use of an adjuvant. The immunized mice showed significant protection against a lethal JEV challenge.

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Figures

FIG. 1.
FIG. 1.
Map of the expression vector pVexCP. The E. coli expression vector pVexCP was made by cloning the modified DNA encoding JGMV CP downstream of the bacteriophage T7 promoter between the NdeI and the BamHI sites of vector pVex11. The unique PstI site, 42 nucleotides upstream of the CP stop codon, is marked. JEV peptide encoding DNA was fused directly to CP cDNA by swapping the PstI and the BamHI fragment. In the case of the linker-mediated fusion of JEV peptide to CP, the linker-encoding DNA replaced the PstI and the BamHI fragment of pVexCP and the JEV peptide encoding DNA was then placed between the BamHI and the HindIII sites. All fusion constructs were sequenced to ensure the presence of the desired peptide- and/or the linker-encoding DNA in the correct reading frame.
FIG. 2.
FIG. 2.
Synthesis in E. coli of JGMV CP fusion proteins containing JEV peptide. Expression of various plasmid constructs containing fusion of CP DNA with DNA-encoding JEV peptides A, B, C, and D, with or without the linker, was studied in E. coli BL21. The cell lysates were separated on a SDS-12% PAGE gel. Shown above is the Coomassie blue-stained gel. Lane 1, protein size markers in kilodaltons (indicated at the left); lane 2, E. coli lysate containing CP; lane 3, E. coli lysate containing CP-A; lane 4, E. coli lysate containing CP-B; lane 5, E. coli lysate containing CP-C; lane 6, E. coli lysate containing CPL; lane 7, E. coli lysate containing CPL-A; lane 8, E. coli lysate containing CPL-B; lane 9, E. coli lysate containing CP-LC; lane 10, E. coli lysate containing CPL-D.
FIG. 3.
FIG. 3.
VLP formation in E. coli by JGMV CP fused with JEV peptide A. pVexCP-A transformed E. coli BL21 cells were grown and induced for the CP-A synthesis with 1 mM IPTG at 37°C. (A to C) Transmission electron micrographs of a section of E. coli cell synthesizing JGMV CP-A seen at ×26,000 magnification (A), a section of E. coli cell synthesizing JGMV CP-A seen at ×52,000 magnification (B), and an E. coli cell lysate containing JGMV CP-A seen at ×105,000 magnification (C). Long, flexuous VLPs are visible in E. coli cells and in the cell lysate.
FIG. 4.
FIG. 4.
Anti-peptide antibody response in mice. Mice were immunized with the recombinant VLPs presenting JEV peptides. Three weeks later a booster immunization was given that was followed by another booster given 4 weeks postimmunization. Mice were bled at various time points. Serum samples were diluted 1:50, and anti-peptide antibodies were analyzed by ELISA with the cognate peptide. For the ELISA of the bleeds obtained from mice immunized with CPL-D, peptide A was used as the capture antigen. The mean ELISA absorbance values for different groups of mice are shown. The solid bars represent preimmunization bleeds, the dotted bars represent bleeds obtained 3 weeks postimmunization, the open bars represent bleeds obtained 1 week after the first booster, and the horizontally lined bars represent bleeds obtained 1 week after the second booster.
FIG. 5.
FIG. 5.
Anti-JEV E protein antibody response in mice. Mice were immunized with the recombinant VLPs presenting JEV peptides. Three weeks later a booster immunization was given that was followed by another booster given 4 weeks postimmunization. Mice were bled at various time points. Serum samples were diluted 1:50, and anti-E antibodies were analyzed by ELISA with the E protein of JEV for the antibody capture. The mean ELISA absorbance values for different groups of mice are shown. The solid bars represent preimmunization bleeds, the dotted bars represent bleeds obtained 3 weeks postimmunization, the open bars represent bleeds obtained 1 week after the first booster, and the horizontally lined bars represent bleeds obtained 1 week after the second booster.
FIG. 6.
FIG. 6.
Endpoint ELISA titers of mice sera. Mice were immunized with the recombinant VLPs presenting JEV A peptide as CP-A or CPL-A. Three weeks later, a booster immunization was given that was followed by another booster given 4 weeks postimmunization. Mice were bled at various time points. Serum samples were diluted 1:50, and anti-peptide (A), anti-E (B), and anti-JEV (C) antibodies were assayed by ELISA with peptide A, JEV E protein, or JEV virions for the antibody capture, respectively. The upper panels show the mean ELISA optical densities (O.D.). The serum samples were serially diluted and assayed for the ELISA endpoint titers. The reciprocal of the highest sera dilution that gave an optical density at least twice of that given by the negative control was considered the ELISA endpoint. The lower panels show the mean endpoint titers. The solid bars represent preimmunization bleeds, the diagonally lined bars represent bleeds obtained 3 weeks postimmunization, the horizontally lined bars represent bleeds obtained 1 week after the first booster, and the open bars represent bleeds obtained 1 week after the second booster.
FIG. 7.
FIG. 7.
Mouse survival after challenge. Mice immunized with VLPs containing CP-A, CPL-A, or CP were challenged 2 weeks after the second booster dose with 10 50% lethal doses of JEV given intracerebrally. Mice were observed for mortality. The percentage of surviving mice at a given time point is shown. ○, Mice immunized with VLPs containing CP-A; ▵, mice immunized with VLPs containing CPL-A; ▿, mice immunized with VLPs containing CP; □, unimmunized mice.
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