FIG 3.
Naturally occurring amino acid variation affects the immunogenicity of DENV-2 DNA vaccines. (A) Total IgG antibody responses in mice injected with different DENV-2 DNA vaccines were determined by a standard Ag-capture ELISA 4 weeks following the last immunization. **, P = 0.0057; ***, P = 0.0007, ****, P < 0.0001. (B) The total IgG ELISA titer is positively and significantly correlated with the VLP secretion titer, as determined by a regression correlation analysis (r = 0.9511; P = 0.0129). (C) Percent blocking of an HRP-labeled anti-prM MAb (155-49) by sera from mice vaccinated with different DENV-2 genotypes containing antibodies to VLPs of the different DENV-2 genotypes determined by an epitope-blocking ELISA. *, P < 0.05. (D) The ratio of uncleaved prM and E among the VLPs of the different DENV-2 genotypes was measured by ELISA and is expressed as a percentage relative to that for immature pVD2-Asian 1 VLPs. *, P = 0.0366. (E) Cross-neutralization activity of antisera elicited by vaccines containing the different DENV-2 genotypes against a set of viruses of different DENV-2 genotypes determined by FRμNT. Nt50 titers are presented as a heatmap generated using the web tool of the HIV sequence database. Lower Nt50 values are red, and the colors progress to orange and then yellow to bright yellow at higher values. GMT, geometric mean titer. *, P < 0.05; ****, P < 0.0001. (F) The total IgG ELISA titer is negatively correlated with the Nt50 titer upon regression correlation analysis (r = 0.5673; P = 0.3186). (G) Ratio of Nt50 titers to DENV-2-specific ELISA titers as a measure of the functional neutralizing activity of the elicited immune sera. ELISA data are means ± SEMs of two independent repeat experiments. **, P = 0.0038; ***, P = 0.003; ****, P < 0.0001. Nt50 titers are expressed as the mean ± 95% CI. Means were analyzed by one-way ANOVA (P < 0.05), and Holm-Sidak's posttest significance is indicated with asterisks.