FIG. 9.

In vivo and in vitro neutralization of AAV-DJ and wild-type AAVs. (A) Mice (n = 4 per group) passively immunized with IVIG (4 or 20 mg) were injected with hFIX-expressing AAV. Plasma hFIX levels per virus and time point are shown as percentages of corresponding levels in control mice (those receiving PBS instead of IVIG). (B) Mice (n = 4 per group) immunized with the higher IVIG dose were also injected with the AAV-DJ HBD mutants. AAV-2, AAV-9, and AAV-DJ were included as controls. hFIX expression from the HBD mutants was marginal, comparable to that from AAV-2. (C) Mice (n = 4 per group) were injected with PBS or 10¹¹ particles of hAAT-expressing AAV-2, AAV-8, AAV-9, or AAV-DJ (x axis), and 3 weeks later, they were reinjected with 10¹¹ particles of hFIX-expressing viruses (5 × 10¹¹ for the least efficient AAV-2, due to the enzyme-linked immunosorbent assay detection limit of ∼10 ng/ml). Shown are stable hFIX levels for each group as measured 6 weeks after the second injection. (D) Sera were taken from the mice described in the legend to panel C at the time of reinjection (bars H [higher dose]), as well as from a parallel group injected with a lower dose (bars L) of 2 × 10¹⁰ particles. Titers of neutralizing antibodies (NAb) against the wild-type AAVs or AAV-DJ were determined as detailed in Materials and Methods. pi, postinjection.