Immunogenicity of hFIX variants. Mice were either made tolerant to hFIX-WT by nonviral gene transfer into the liver or were left naive as controls. (A) Graph showing hFIX-WT expression in groups of BALB/c mice over time. The different arrows indicate the vaccination events with variants T (n = 5), ITV (n = 6), and WT (n = 5) and the time point for Ab measurement. For vaccination leading to the induction of Abs, we injected 500 μg/mouse of pCDNA3.1.CMV-hFIX-WT, hFIX-T, or hFIX-ITV into both tibialis anterior muscles. The procedure was repeated twice at the indicated time points to boost Ab development. Four weeks after the last immunization boost, Ab titers were measured by anti-hFIX Ig-specific ELISAs for IgG1 (B) and IgG2a (C). Depending on the treatment group, hFIX-T, hFIX-ITV, or hFIX-WT was used for coating to allow detection of epitope-specific Abs. (D) For the Bethesda assay, we used an hFIX variant (ITV) that included all tested amino acid exchanges for the detection of functional epitope-specific Abs.