Humoral responses to hFIX-Padua following challenge. Ten to 15 weeks after administration of 5 × 10¹⁰ vg/kg AAV8-hFIX-R338L or AAV-hFIX-WT, HB mice were assessed for development of anti-hFIX IgG antibodies following subcutaneous challenges with 2 μg per mouse recombinant hFIX-WT or hFIX-Padua with protein alone, protein with CFA, and protein with IFA. Untreated HB mice challenged with protein alone served as a positive control. **P < .01; ***P < .001.