Characterization of AAV-Specific Affinity Ligands: Consequences for Vector Purification and Development Strategies

Affinity-based purification of adeno-associated virus (AAV) vectors has replaced density-based methods for vectors used in clinical settings. This method utilizes camelid single-domain antibodies recognizing AAV capsids. These include AVB Sepharose (AVB) and POROS CaptureSelect affinity ligand for AAV8 (CSAL8) and AAV9 (CSAL9). In this study, we utilized cryo-electron microscopy and 3D image reconstruction to map the binding sites of these affinity ligands on the capsids of several AAV serotypes, including AAV1, AAV2, AAV5, AAV8, and AAV9, representing the range of sequence and structure diversity among AAVs. The AAV-ligand complex structures showed that AVB and CSAL9 bound to the 5-fold capsid region, although in different orientations, and CSAL8 bound to the side of the 3-fold protrusion. The AAV contact residues required for ligand binding, and thus AAV purification, and the ability of the ligands to neutralize infection were analyzed. The data show that only a few residues within the epitopes served to block affinity ligand binding. Neutralization was observed for AAV1 and AAV5 with AVB, for AAV1 with CSAL8, and for AAV9 with CSAL9, associated with regions that overlap with epitopes for neutralizing monoclonal antibodies against these capsids. This information is critical and could be generally applicable in the development of novel AAV vectors amenable to affinity column purification.