Immune response to hFVIII in vector-treated hemophilia A mice without immunotolerization. (A) Hemophilia A mice were given a high-pressure tail-vein injection of 200 μg pSBTF8 with 80 μg of UB-SB10 (▦) or a luciferase plasmid (▴;n = 4/group), and plasma was subsequently assayed for hFVIII by ELISA. (B) Same as in A, except n = 3/group, and hFVIII activity in plasma was assayed by COATEST. (C) Equal volume of pooled normal human plasma was mixed with plasma from vector-treated mice (in panel B) and incubated for 2 hours at 37°C. Bethesda units were determined by dilution of plasma, whereby each unit corresponded to a 50% reduction in hFVIII activity measured by COATEST. Circulating hFVIII activity declined as anti-hFVIII antibody level increased, thus showing a humoral immune response to hFVIII. (A-C, error + SD).