AAV6 K531 serves a dual function in selective receptor and antibody ADK6 recognition

Abstract

Adeno-associated viruses (AAVs) are being developed as vectors for the treatment of genetic disorders. However, pre-existing antibodies present a significant limitation to achieving optimal efficacy for the AAV gene delivery system. Efforts aimed at engineering vectors with the ability to evade the immune response include identification of residues on the virus capsid important for these interactions and changing them. Here K531 is identified as the determinant of monoclonal antibody ADK6 recognition by AAV6, and not the closely related AAV1. The AAV6-ADK6 complex structure was determined by cryo-electron microscopy and the footprint confirmed by cell-based assays. The ADK6 footprint overlaps previously identified AAV antigenic regions and neutralizes by blocking essential cell surface glycan attachment sites. This study thus expands the available repertoire of AAV-antibody information that can guide the design of host immune escaping AAV vectors able to maintain capsid functionality.

High-Resolution Structural Characterization of a New Adeno-associated Virus Serotype 5 Antibody Epitope toward Engineering Antibody-Resistant Recombinant Gene Delivery Vectors

“…The 60 VPs create the characteristic features of the AAV capsids with cylindrical channels at the 5-fold axes, protrusions surrounding the 3-fold axes, and depressions at the 2-fold axes and surrounding the 5-fold channels that are separated by "walls," termed 2/5-fold walls. Variable regions (VRs) I to IX (VR-I to VR-IX), defined for the AAVs based on sequence and structure differences clustered at or around these capsid features, result in phenotypic differences, such as in receptor attachment and antigenicity, between the AAV serotypes (5,(8)(9)(10)(11)(12)(13)(14)(15).…”

“…In order to identify the antigenic regions, cryo-electron microscopy (cryo-EM) and threedimensional (3D) image reconstruction of AAV capsids in complex with antibodies has been used. This approach has led to the mapping of the binding sites of several monoclonal antibodies (MAbs) to multiple AAV serotypes, e.g., ADK1a, ADK1b, 4E4, and 5H7 to AAV1 (9,11), A20 and C37-B to AAV2 (9,23), ADK5a, ADK5b, and 3C5 to AAV5 (9,11), ADK6 to AAV6 (13), and ADK8 to AAV8 (10). Most of these antibodies efficiently neutralize infectivity by the respective AAV vector.…”

“…The resolutions of the reconstructed capsid-antibody complexes ranged from 6.7 to 23 Å, allowing the mapping of a "footprint" indicating a cluster of capsid surface residues that are potentially involved in the capsid-antibody interaction. In the case of AAV6 and AAV8, single-residue substitutions or loop swap mutations successfully restored infectivity in the presence of the neutralizing antibodies (10,13).…”

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

“…AAV virus-like particles (VLPs) were expressed using a recombinant baculovirus expressing the VPs of the desired AAV serotype. VLPs were purified as described before 48 and dialyzed into 20 mM Tris-HCl and 250 mM NaCl (pH 7.5) for AAV1, AAV2, AAV5, and AAV8, and 20 mM Tris, 350 mM NaCl, and 2 mM MgCl 2 (pH 7.4) for AAV9. Virus purity was confirmed by SDS-PAGE and negative stain EM.…”

Biophysical, Biochemical, and Cell Based Approaches Used to Decipher the Role of Carbonic Anhydrases in Cancer and to Evaluate the Potency of Targeted Inhibitors

“…To date, no CA structures have been solved using this method. This is because Cryo-EM enables the study of specimens larger than 150 kDa including viruses, small organelles, and macromolecular biological complexes as well as molecular interactions in supramolecular assemblies or machines [ 112 – 114 ]. The average molecular weight of CAs in the presence or absence of inhibitors is ~30 kDa [ 115 ].…”