compared to IgG1 and only detectable on day 12 after the first immunization (PP ratio of IgG1:IgG2A = 2.8:1). On the day before RVFV challenge (IgG2A: average = 23.4 PP, standard deviation = 4.2 PP, OD405 nm = 0.22), a weak IgG2A response was still detectable(PP ratio of IgG1:IgG2A = 5.6:1) (Figure 1). Dynamics of gene expression and virus load in livers and spleens of immunized and control mice The normalized expression of IL-10, IFNc and IFNb was analyzed at 3, 6, 12, 24, 72 and 120 hours post RVFV infection in liver and spleen tissue of recNP immunized, and non-immunized adjuvant and PBS control mice. The results are shown in Figure 2a–f. The expression of IL-10 was upregulated with statistical significance (4.5 fold, p,0.01) in adjuvant control mice but normal in recNP immunized mice at 3 hours p.i. in the liver (Figure 2a). At 72 and 120 hours p.i., however, IL-10 expression was massively upregulated in the adjuvant (72 hours, 808.3 fold, p = 0.033; 120 hours, 55.0 fold, p,0.01) and PBS control groups (72 hours, 243.3 fold, p,0.01; 120 hours, 99.3 fold, p = 0.034), and upregulated to a lesser extent in immunized mice (72 hours, 24.4 fold, p,0.01; 120 hours, 24.3 fold, p = 0.033) in the liver Protective Anti-RVFV NP Responses in Mice (Figure 2a). In the spleen expression of IL-10 was only upregulated significantly at 72 hours p.i. in control mice (adjuvant 46.0 fold, p,0.01; PBS 55.0 fold, p,0.01) (Figure 2b). The expression of IFNc was normal in all mice livers at 3 hours, but upregulated in all groups at 6 hours (recNP immunized, 17.2 fold, p,0.01; adjuvant control, 11.7 fold, p,0.01; PBS control, 9.3 fold, p,0.01), 72 hours (recNP immunized, 8.4 fold, p = 0.03; adjuvant control, 5.8 fold, p = 0.03; PBS control, 4.4 fold, p = 0.03) and 120 hours p.i. (recNP immunized, 23.4 fold, p = 0.03; adjuvant control 224.0 fold, p,0.01; PBS control 237.0 fold, p = 0.03) (Figure 2c). Expression of IFNc remained unaltered in spleen tissue (Figure 2d). At 3 hours post infection (p.i.) the expression of the IFNb gene was significantly upregulated in the livers (7.7 fold, p = 0.03) and spleens (12.0 fold, p = 0.03) of recNP immunized mice, whereas expression was normal in adjuvant control and PBS control groups (Figure 2e–f). Expression of the gene remained upregulated in liver of immunized mice until 12 hours p.i., after which it waned (Figure 2e–f). In adjuvant control mice, IFNb was massively upregulated at 72 hours p.i. in liver (2171.7 fold, p = 0.03) and spleen (542.3 fold, p,0.01), with similar results obtained in PBS control mice liver (2524.1 fold, p = 0.02) and spleen (510.3 fold, Figure 2. (a–f). Fold changes in expression of IL10, IFNc and IFNb genes in tissues of mice after RVFV infection. RecNP immunized mice (n = 3 per time point) are indicated by solid black bars, adjuvant control mice (n = 3 per time point) by grey bars and PBS control mice (n = 3 per time point) by white bars. The horizontal dotted lines indicate the cut-off values for upregulation (+2) or downregulation (22). The asterisk (*) indicates where the P-value is smaller than or equal to 0.05 (statistically significant results). Standard error values are indicated by the error bars. Note the differences in the Y-axis scales. The following time points are indicated: 3, 6, 12, 24, 72 and 120 hours. doi:10.1371/journal.pone.0025027.g002 PLoS ONE | www.plosone.org 3 September 2011 | Volume 6 | Issue 9 | e25027