Figure 3.

Schematic representation and immunogenicity of tested DNA vaccines encoding DENV2 NS1, NS3, or NS5. Gene sequences encoding the nonstructural proteins 1, 3, and 5 from DENV 2 (strain JHA1) were used to develop the DNA vaccines pNS1, pNS3, and pNS5, respectively (A). BHK cells were transfected with each plasmid encoding NS1, NS3, or NS5, and protein expression was detected by anti-NS1 4F6 mab (B), anti-NS3 polyclonal antibody (C), or anti-NS5 GT361 mab (D), followed by incubation with anti-mouse fluorescein-conjugated goat IgG. Splenocytes from mice (n = 5) immunized with two doses of each DNA vaccine were obtained two weeks after the last dose and stimulated with a pool of peptides from NS1, NS3, or NS5, and the result was assessed by IFN-γ ELISpot analysis (E). Two weeks after the last dose of each vaccine, mice were bled, and sera were tested for the presence of DENV2 NS1, NS3h, or NS5-reactive IgG by ELISA (F–H respectively), using the recombination versions of DENV 2 full-length NS1 and NS5, as well the domain helicase of NS3. Statistical analyses were performed by a nonparametric Mann-Whitney test, where *P < 0.05 and **P < 0.01.