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

Abstract

Adeno-associated virus serotype 5 (AAV5) is being developed as a gene delivery vector for several diseases, including hemophilia and Huntington’s disease, and has a demonstrated efficient transduction in liver, lung, skeletal muscle, and the central nervous system. One limitation of AAV gene delivery is preexisting neutralizing antibodies, which present a significant challenge for vector effectiveness in therapeutic applications. Here, we report the cryo-electron microscopy (cryo-EM) and image-reconstructed structure of AAV5 in complex with a newly generated monoclonal antibody, HL2476, at 3.1-Å resolution. Unlike other available anti-AAV5 capsid antibodies, ADK5a and ADK5b, with epitopes surrounding the 5-fold channel of the capsid, HL2476 binds to the 3-fold protrusions. To elucidate the capsid-antibody interactions, the heavy and light chains were sequenced and their coordinates, along with the AAV5 viral protein, assigned to the density map. The high resolution of the complex enabled the identification of interacting residues at the 3-fold protrusions of the capsid, including R483, which forms two hydrogen bonds with the light chain of HL2476. A panel of AAV5 variants was generated and analyzed by native dot immunoblot and transduction assays. This identified variants with antibody escape phenotypes that maintain infectivity. IMPORTANCE Biologics based on recombinant AAVs (rAAVs) are increasingly becoming attractive human gene delivery vehicles, especially after the approval of Glybera in Europe and Luxturna in the United States. However, preexisting neutralizing antibodies against the AAV capsids in a large percentage of the human population limit wide-spread utilization of these vectors. To circumvent this problem, stealth vectors must be generated that are undetectable by these antibodies. This study details the high-resolution characterization of a new antigenic region on AAV5, a vector being developed for numerous delivery applications. The structure of AAV5 complexed with HL2476, a novel antibody, was determined by cryo-EM to 3.1-Å resolution. The resolution of the density map enabled the identification of interacting residues between capsid and antibody and the determinants of neutralization. Thus, the information obtained from this study can facilitate the generation of host immune escape vectors.

Comparative Analysis of the Capsid Structures of AAVrh.10, AAVrh.39, and AAV8

“…A similar density to that extending the channel at the 5-fold axis in the empty AAV8 and AAVrh.39 structures has been previously reported for bocaparvoviruses (BoVs) VLPs and AAV5 VLPs complexed an FAb (60)(61)(62). The first N-terminal residue in the ordered VP region for all currently known parvovirus VP structures is located at the base of the 5-fold channel (63).…”

“…Recombinant AAV production and purification. Recombinant AAV8, AAVrh.10, and AAVrh.39 vectors, with a packaged luciferase gene, were produced by triple transfection of HEK293 cells, utilizing pTR-UF3-Luciferase, pHelper (Stratagene), and either pXR8, pAAV2/rh.10, or pAAV2/rh.39; purified by AVB Sepharose affinity chromatography; and concentrated as previously described (60).…”

The Structure of an AAV5-AAVR Complex at 2.5 Å Resolution: Implications for Cellular Entry and Immune Neutralization of AAV Gene Therapy Vectors

“…One can speculate that the lack of transduction inhibition with an antibody that occludes receptor-binding could result from receptor sites that are left unblocked, or that antibody saturation of the virus requires a less favorable 3C5 configuration with which AAVR can compete dynamically for binding. The hybridoma-derived neutralizing antibody H2476 had been subject to negative selection for affinity to nanobody AVB, thereby increasing the likelihood of recognizing an epitope near the three-fold symmetry axis [36]. Its footprint overlaps only slightly with that of PKD1 ( Figure 5B,C), but, if coordinates for H2476 were available, greater steric conflict between the bound proteins might The hybridoma-derived neutralizing antibody H2476 had been subject to negative selection for affinity to nanobody AVB, thereby increasing the likelihood of recognizing an epitope near the three-fold symmetry axis [36].…”

“…The hybridoma-derived neutralizing antibody H2476 had been subject to negative selection for affinity to nanobody AVB, thereby increasing the likelihood of recognizing an epitope near the three-fold symmetry axis [36]. Its footprint overlaps only slightly with that of PKD1 ( Figure 5B,C), but, if coordinates for H2476 were available, greater steric conflict between the bound proteins might The hybridoma-derived neutralizing antibody H2476 had been subject to negative selection for affinity to nanobody AVB, thereby increasing the likelihood of recognizing an epitope near the three-fold symmetry axis [36]. Its footprint overlaps only slightly with that of PKD1 ( Figure 5B,C), but, if coordinates for H2476 were available, greater steric conflict between the bound proteins might be apparent further away from the virus surface.…”

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

“…Vector genome-containing AAVs were produced by triple transfection of HEK293 cells, utilizing pHelper (Stratagene, San Diego, CA, USA), pTR-UF3-luciferase, and either pXR1, pXR2, pXR5, pXR8, or pXR9 as previously described. 47 The transfected cells were harvested 72 h after transfection. Cells were pelleted at 2,000 × g for 5 min and resuspended in 1× TD buffer (1× PBS supplemented with 1 mM MgCl 2 and 2.5 mM KCl) and subjected to three freeze-thaw cycles.…”