hFVIII activity after LV.pF8.FVIII delivery in HA mice previously immunized with hFVIII. (A) Graphic representation of hFVIII activity (red line) and anti-FVIII antibody (Ab) titer (blue line) trend of C57BL/6 HA injected with LV.pF8.FVIII after immunization (n = 4). (B) Bethesda assay showing formation of high titer inhibitors (≥20 BU) in all immunized mice. (C) Graph showing quantification of anti-FVIII isotypes IgG1, IgG2a, and IgG2b in plasma of FVIII-immunized and subsequently injected with LV.pF8.FVIII. High titer of IgG1 and IgG2a were predominantly detectable in all mice. Bleeding assay (D) and bleeding time (E) showing a significant phenotypic correction of HA treated mice. *P ≤ .0001.