Figure 1.

Geometric mean frequencies (GMFs) of (A) gE-specific CD4⁺ T cells and (B) ratios of GMFs from different adjuvanted-vaccine groups. Spleens (Experiment 1, N=8 and Experiment 2, N = 11; spleens pooled from 2 mice) were sampled at 30 d after the second vaccine dose (30dPII). The frequency of gE-specific CD4⁺ T cells was calculated as a percentage of cytokine-positive CD4⁺ T cells divided by all CD4⁺ T cells. Error bars represent 95% confidence intervals. In Experiments 1 and 2, the frequencies of antigen-specific CD4⁺ T cells in the NaCl group were either close to or below the cut-off for the assay (GMFs were 0.3% and 0.05%, respectively). In (B), horizontal gray reference line indicates a ratio = 1, and asterisks indicate significant differences from 1 (** p<0.01; *** p<0.001). Antigen-specific T cells were evaluated in splenocyte-restimulation cultures as described previously,¹³ but with some modifications. Briefly, splenocyte cultures (110⁶ cells per well of 96-well plate) were prepared from spleens of 2 mice and were incubated for 2 hours in the presence of gE peptides spanning the complete gE protein (6315-mer peptides, 11 amino-acid overlap) and then incubated ˜18 hours in the presence of brefeldin A. Subsequently, the cells were stained with fluorescent-monoclonal antibodies specific for CD4 and after permeabilization, for intracellular-cytokines IL-2 and IFN-γ. All antibodies were obtained from BD Biosciences, Belgium. Flow cytometry was performed using LSR II Facs (BD Biosciences, Belgium) and analyzed using FlowJo software (FlowJo, LLC, OR, USA). Statistical calculations were based on an analysis of variance with 2 factors (vaccine group, experiment) on log₁₀ values using a heterogeneous variance model (i.e., identical variances were not assumed for the different levels of the factor). Estimates of the geometric-mean ratios between groups and their 95% confidence intervals (CI) were obtained using back-transformation of log₁₀ values. Adjustments for multiple testing were performed using Tukey's method. All analyses were performed using SAS software (Version 9.2, SAS Institute Inc., NC, USA).