Successful Transduction with AAV Vectors after Selective Depletion of Anti-AAV Antibodies by Immunoadsorption

Figure 1. Depletion of Anti-AAV9 Antibodies from an IVIG Solution by Immunoadsorption with AAV9 Sepharose Beads (A) Schematic depiction of immunoadsorption of anti-AAV9 antibodies. To deplete anti-AAV9 antibodies from a solution with human IVIG (intravenous immunoglobulin), was incubated the solution with Sepharose beads with covalently coupled AAV9 virions (AAV9-beads). (B) After incubation with IVIG solution, the beads were washed with PBS and the anti-AAV antibodies were eluted from the AAV9-beads by incubation with a low pH buffer. After elution of the anti-AAV antibodies, the beads were washed with PBS. The binding/elution procedure was repeated twice. Anti-AAV9 antibodies in the eluates were detected by Western blot with neutralized eluates as a primary antibody. The AAV9 capsid proteins (VP1, VP2, and VP3) were detected with an HRP-anti-human-IgG secondary antibody and ECL. The same matrix was reused three times without evidence of loss of binding capacity. (C) As in (B), but with BSA-beads. (D) Western blot experiment showing the presence of immunoglobulin heavy chain (HC) and light chain (LC) in the AAV9-beads eluates. (E) The total amount of antibodies remained unchanged after…

Figure 2. Analysis of Total Anti-AAV9 Antibody Titers in the Supernatants and Eluates of the Experiments Described in Figure 1 (A) Total amounts of anti-AAV9 antibodies in the supernatants (Figure 1) were determined by ELISA. (B) Total amounts of anti-AAV9 antibodies in the eluates (Figure 1) were determined by ELISA. The data are depicted as the mean ± standard error (n = 3).

Figure 3. Binding Capacity for Anti-AAV9 Antibodies 50 μL AAV9-beads or BSA-beads were incubated with increasing concentrations of IVIG. The coupled antibodies were eluted and the amount of anti-AAV9 antibody bound was determined by ELISA. The data are depicted as the mean ± standard error (n = 3).

Figure 4. IVIG but Not Supernatants of IVIG Solutions Incubated with AAV9-Beads Inhibit Transduction of HEK293T Cells with AAV9-Luc (A) Anti-AAV9 antibodies were depleted from human IVIG solutions by incubation with AAV9-beads (BSA-beads served as a control). The same beads were used for three rounds of depletion (see Figure 1 and text). (B) 2-fold dilutions of the supernatants of the IVIG solutions with AAV9-beads or BSA-beads were used to detect transduction of HEK293T cells by an AAV9 vector-encoding luciferase (AAV9-Luc). For details, see Materials and Methods section. (C) After the incubation with the AAV9-beads or BSA-beads, bound anti-AAV9 antibodies were eluted with low pH buffer. Neutralized supernatants were then used to detect the presence of neutralizing antibodies against AAV9 as described in (B). The transduction efficiency is expressed as percent of no-serum control samples. The data are depicted as the mean ± standard error (n = 3).

Figure 5. Depletion of Anti-AAV Antibodies in Human Serum Samples by Immunoadsorption In vitro depletion of anti-AAV9 antibodies from 5 human sera with neutralizing titers of 1/4, 1/8, 1/16, 1/32, and 1/128, respectively. Incubation and elution was performed as described in Figure 1. Neutralizing antibody titers were determined as in Figure 2, and total anti-AAV9 antibodies amounts were quantified by ELISA. The data are depicted as the mean ± standard error (n = 3). Western blot was used to confirm that the antibodies eluted from AAV9-beads were specific for AAV9 capsid proteins (see also Table S1).

Figure 6. Hemapheresis and Immunoadsorption with AAV9 Beads Depletes Anti-AAV9 Antibodies from the Blood of Passively Immunized Rats (A) Experimental scheme of the depletion protocol. At day 0, catheters were implanted into the jugular vein of rats that were passively immunized with anti-AAV9 rat serum. On day 1, rats underwent three rounds of hemapheresis and immunoadsorption with AAV9 beads. Blood was drawn immediately before the first hemapheresis session (baseline) and immediately after each round of hemapheresis. Immediately after the last hemapheresis session, 8 × 1012 vg/kg of AAV9-Luc was injected. (B) Sera from the blood samples described in (A) were used for neutralization assays (see Figure 2). (C) Total anti AAV9 antibodies were determined by ELISA (for details, see Materials and Methods section). The data are expressed as the percentage of total-anti AAV9 antibodies at baseline.

Figure 7. Depletion of Anti-AAV9 Antibodies by Hemapheresis and Immunoadsorption from the Blood of Passively Immunized Rats Allows the Successful In Vivo Transduction with AAV9-Luc (A) Timeline of experiment. (B) Expression of luciferase determined by in vivo imaging (day 21; left) and luciferase expression measured in vitro (day 28; right) in tissue of the heart, liver, and skeletal muscle harvested at the day of sacrifice. The average of background luminescence in tissues of uninjected rats (n = 4) was subtracted from the values obtained in animals that were injected with AAV-Luc. The neutralizing titer of immunized animals or of immunized animals before hemapheresis and immunoadsorption was 1/8. Naive animals (n = 10), immunized (n = 6), immunized plus hemapheresis and immunoadsorption (n = 5). NC, not calculable because the values of immunized rats were below zero RLU/mg (relative luciferase units/mg protein) (C) As in (B) but the neutralizing titer of immunized animals or of immunized animals before hemapheresis and immunoadsorption was 1/16. Naive animals (n = 10), immunized (n = 5), and immunized plus hemapheresis and immunoadsorption (n = 5). Boxes correspond to upper and lower…