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

Area of Science:

• Biochemistry
• Structural Biology
• Virology

Background:

• Affinity purification using camelid single-domain antibodies is preferred over density-based methods for adeno-associated virus (AAV) vectors in clinical applications.
• Key affinity ligands include AVB Sepharose (AVB), POROS CaptureSelect for AAV8 (CSAL8), and for AAV9 (CSAL9).

Purpose of the Study:

• To map the binding sites of AVB, CSAL8, and CSAL9 on various adeno-associated virus (AAV) serotypes using cryo-electron microscopy.
• To analyze AAV contact residues essential for ligand binding and neutralization.
• To assess the potential for developing novel AAV vectors with enhanced affinity purification capabilities.

Main Methods:

• Utilized cryo-electron microscopy (cryo-EM) and 3D image reconstruction to determine the structures of AAV-ligand complexes.
• Investigated binding sites on AAV serotypes AAV1, AAV2, AAV5, AAV8, and AAV9.
• Analyzed AAV contact residues and evaluated ligand-mediated neutralization of viral infection.

Main Results:

• Determined structures of AVB, CSAL8, and CSAL9 bound to AAV capsids, revealing specific binding orientations at the 5-fold and 3-fold protrusion regions.
• Identified key AAV residues critical for ligand binding and demonstrated that only a few residues within epitopes block binding.
• Observed neutralization of AAV infection by AVB (AAV1, AAV5), CSAL8 (AAV1), and CSAL9 (AAV9), correlating with epitopes of neutralizing antibodies.

Conclusions:

• Structural insights into AAV-ligand interactions provide a basis for optimizing affinity purification processes.
• Understanding epitope-ligand interactions is crucial for developing effective AAV purification and neutralization strategies.
• This knowledge is broadly applicable for engineering AAV vectors with improved affinity column purification characteristics.