Epitope mapping of human anti-adeno-associated virus type 2 neutralizing antibodies: Implications for gene therapy and virus structure

Recombinant adeno-associated virus type 2 (AAV) is a common vector used in human gene therapy protocols. We characterized the humoral immune response to AAV and observed that 80% of normal human subjects have anti-AAV antibod ies and that 18% have neutralizing antibodies. To analyze the effect of neu tralizing antibodies on AAV readministration, we attempted to deliver recom binant AAV expressing human factor IX (AAV-hFIX) intraportally into the liv ers of mice which had been preexposed to AAV and shown to harbor a neutrali zing antibody response, While all naive control mice expressed hEIX followi ng administration of AAV-hFIX, none of the mice with preexisting immunity e xpressed hFIX, even after transient immunosuppression at the time of the se cond administration with anti-CD4 or anti-CD40L antibodies. This suggests t hat preexisting immunity to AAV, as measured by a neutralizing antibody res ponse, may limit AAV-mediated gene delivery. Using human sera in an enzyme- linked immunosorbent assay for AAV and a capsid peptide scan library to blo ck antibody binding, we mapped seven regions of the AAV capsid containing i mmunogenic epitopes. Using pools of these peptides to inhibit the binding o f neutralizing antibodies, we have identified a subset of six peptides whic h potentially reconstitute a single neutralizing epitope. This information may allow the design of reverse genetic approaches to circumvent the preexi sting immunity that can be encountered in some individuals.