Figure 3.

NLC Modifications That Modulate Immune Responses

(A) SEAP expression in peripheral serum 3 days after C57BL/6 mice (n = 3/group) were injected IM with 10% sucrose (mock) or 100 ng of SEAP rvRNA unformulated (naked) or complexed with CNE (containing Span 85) or NLCs containing Span 60, 80, or 85. Each data point is plotted as well as their mean ± SD. (B) Eighty percent reduction neutralizing antibody titers, measured by PRNT₈₀, 14 days after C57BL/6 mice (n = 5/group) were injected IM with 10% sucrose (mock) or 100 ng of ZIKV rvRNA unformulated (naked) or complexed with CNE (containing Span 85) or NLCs containing Span 60, 80, or 85. Each data point is plotted as well as their geometric mean ± SD. (C) ZIKV neutralization with serial dilutions of serum harvested from C57BL/6 mice (n = 5/group) 14 days after IM vaccination with 100 ng of ZIKV rvRNA complexed with NLCs or CNEs containing Span 60 or 85. Each serum dilution is plotted as mean ± SD, and a curve was fitted using a sigmoidal non-linear regression model. (D) Mip-1β concentration, as determined by ELISA, in supernatants of human peripheral blood mononuclear cells (n = 6 donors) harvested 24 hr after incubation with 40 ng ZIKV rvRNA unformulated (naked) or complexed with CNE containing Span 60 or 85 in a squalene emulsion or with NLCs containing Span 60, 80, or 85 and squalene and Dynasan or NLC containing Span 60 and miglyol and Dynasan emulsion. Two technical ELISA replicates per donor were performed, and the mean of each donor is plotted along with the mean ± SD of the six donors. Data in (A) and (B) are representative of three independent experiments. Data in (C) and (D) are representative of two independent experiments. Log₁₀ transform of data in (B) was analyzed by one-way ANOVA with Tukey’s multiple comparison test (NLC Span 60 compared to CNE or NLC Span 85, **p < 0.008; NLC Span 60 compared to NLC Span 80, ***p = 0.0007; CNE compared to NLC Span 80 or 85, not significant). Data in (C) was analyzed by multiple two-way ANOVA with Tukey’s multiple comparison test (at 1/640 serum dilution NLC Span 60 versus CNE Span 85 and CNE Span 60 versus CNE Span 85, *p < 0.05; NLC Span 60 versus CNE Span 60, **p < 0.0001). Data in (D) was analyzed by multiple one-way ANOVA with Tukey’s multiple comparison test (NLC-Span 60-squalene or NLC-Span 85-squalene compared to either naked or CNE-Span 85, **p < 0.005).