FIG. 4.

Presentation of epitopes on the AAV-DJ capsid. Shown at the top are the putative AAV-2 epitope for the monoclonal antibody A20 (a conformational epitope comprising three distinct peptides [see also Fig. 2D]) and the corresponding sequences in AAV-DJ, AAV-8, and AAV-9 (amino acid changes compared to the sequence of AAV-2 are highlighted in red [AAV-DJ] or orange [AAV-8 and AAV-9]). The bottom panels show results from immunofluorescence studies of cells cotransfected with the various helper constructs and an adenoviral helper plasmid (to boost gene expression from the AAV plasmids). The two amino acid changes in AAV-DJ were already sufficient to abolish the binding of the A20 antibody, validating and narrowing down the A20 epitope (80) and thus exemplifying the potential of DNA family shuffling as a reverse-genetics tool. AAV-DJ cross-reacted with both the polyclonal anti-AAV-2 and anti-AAV-8 sera, as expected from its chimeric structure. Similar cross-reactivity with these sera was also observed for wild types 2, 8, and 9. The fact that AAV-DJ, AAV-8, and AAV-9 were detected by the B1 antibody (initially raised against AAV-2 capsid proteins) was not surprising considering the high degree of conservation of its epitope in natural AAVs (see Fig. S4 in the supplemental material). All mono- and polyclonal anti-AAV antibodies were described previously (78, 79), except for the polyclonal rabbit anti-AAV-8 antiserum. 303.9, anti-Rep; B1, anti-VP; αAAV-2, anti-AAV-2 VP serum; A20, anti-AAV-2 capsids; αAAV-8, anti-AAV-8 VP serum.