Figure 4.

Detection of serum neutralizing antibodies, anti-AAV capsid immunoglobulin G (IgG), and anti-luciferase antibodies after intranasal (IN) administration of rAAV-Luc vectors. Mice were administered either 1 × 10¹¹ recombinant adeno-associated virus type 12 (rAAV12)-Luc or rAAV2-Luc vector particles by IN route. (a) Serum neutralizing antibodies (Nab) to AAV12 and AAV2 were measured 4 weeks after initial vector administration as described in the Materials and Methods section. Neutralizing titers were expressed as the reciprocal of the highest serum dilution that still gave 50% transduction with rAAV-Luc vectors compared to naive control serum. No crossreactivity was seen between rAAV12 and rAAV2 antiserum. Error bars represent the mean reciprocal Nab titer to AAV + s.e.m., where n = 7. Statistical comparison was performed by unpaired t-test to compare rAAV12-Luc neutralizing titer with that of the rAAV2-Luc group (****P < 0.0001). (b) Anti-AAV capsid protein IgG titers were also measured 4 weeks post initial rAAV12-Luc and rAAV2-Luc vector administration by ELISA as described in methods. Optical density values of naive serum controls were subtracted from overall values. Error bars represent the mean optical density reading + s.e.m., where n = 7. Statistical comparison was performed by unpaired t-test to compare titer from rAAV12-Luc animals with those of the rAAV2-Luc group (**P < 0.01). (c) Anti-luciferase antibodies were measured in serum samples 2 weeks post boost by direct ELISA. Concentrations of anti-luciferase antibodies were estimated by comparison with a standard curve (anti-luciferase monoclonal antibody, 1 µg–0.001µg/ml range) and expressed in ng/ml. Serum from rAAV12-A/NP- and rAAV2-EGFP-treated animals were included as negative controls for rAAV12-Luc and rAAV2-Luc respectively, and showed no anti-luciferase antibody response. Error bars represent the mean anti-luciferase antibody titer (ng/ml) + s.e.m., where n = 7. Statistical comparison was performed by unpaired t-test to compare corresponding anti-luciferase antibody titers between the two groups (***P < 0.001).