Modulation of antibody formation by oral administration of bioencapsulated FIX in hemophilia B mice. (A) Timeline and schedule for oral gavages (FIX or WT plant material, n = 8 per experimental group), IV FIX administration, and blood collection for antibody assays. (B) Inhibitor titers (in BU/mL plasma). (C) FIX-specific IgG titers (IgG1, IgG2a, and IgG2b (ng/mL). (D) FIX-specific IgE titers (ng/mL) and (E) FIX-specific IgA titers (in ng/mL for circulating IgA and in ng/100 μg total IgA for fecal titers). (F) At the end of the experiment, in vitro splenocyte cultures were established. Cells were stimulated with or without 10 μg/mL FIX antigen for 48 hours, followed by quantitative RT-PCR analysis. “Fold increase” is change in RNA transcripts of FIX vs mock stimulated. All gene expression was compared with that of glyceraldehyde-3-phosphate dehydrogenase. Fold-change was calculated using the 2ΔΔCt quantification method. The dotted horizontal line indicates 1-fold change. (B-F) Average ± standard error of the mean (SEM).