Mapping a Neutralizing Epitope onto the Capsid of Adeno-Associated Virus Serotype 8

Abstract

c Adeno-associated viruses (AAVs) are small single-stranded DNA viruses that can package and deliver nongenomic DNA for therapeutic gene delivery. AAV8, a liver-tropic vector, has shown great promise for the treatment of hemophilia A and B. However, as with other AAV vectors, host anti-capsid immune responses are a deterrent to therapeutic success. To characterize the antigenic structure of this vector, cryo-electron microscopy and image reconstruction (cryo-reconstruction) combined with molecular genetics, biochemistry, and in vivo approaches were used to define an antigenic epitope on the AAV8 capsid surface for a neutralizing monoclonal antibody, ADK8. Docking of the crystal structures of AAV8 and a generic Fab into the cryo-reconstruction for the AAV8-ADK8 complex identified a footprint on the prominent protrusions that flank the 3-fold axes of the icosahedrally symmetric capsid. Mutagenesis and cell-binding studies, along with in vitro and in vivo transduction assays, showed that the major ADK8 epitope is formed by an AAV variable region, VRVIII (amino acids 586 to 591 [AAV8 VP1 numbering]), which lies on the surface of the protrusions facing the 3-fold axis. This region plays a role in AAV2 and AAV8 cellular transduction. Coincidently, cell binding and trafficking assays indicate that ADK8 affects a postentry step required for successful virus trafficking to the nucleus, suggesting a probable mechanism of neutralization. This structure-directed strategy for characterizing the antigenic regions of AAVs can thus generate useful information to help re-engineer vectors that escape host neutralization and are hence more efficacious.

Comparative Analysis of Adeno-Associated Virus Capsid Stability and Dynamics

“…Structural studies of AAV1 and AAV2 with neutralizing antibodies also map antigenic epitopes to the 3-fold region (105). Our proteolytic map also overlies a recent cryo-EM reconstruction of AAV8 in complex with a neutralizing antibody, which highlights the 3-fold region as an important antigenic determinant as well (90). Residues on the 3-fold protrusions of AAV8 also determine receptor binding and transduction efficiency (19,90,91).…”

“…The biological force(s) responsible for driving the biophysical divergence between serotypes is unclear at this time. What is known is that sequence differences in the VRs contribute to phenotypic differences, including receptor attachment, transduction efficiency, and antigenic reactivity (1,3,4,88,90,91,95,96,105). We hypothesize that the observed biophysical differences between serotypes are connected to the specific biology of each serotype.…”

“…Our proteolytic map also overlies a recent cryo-EM reconstruction of AAV8 in complex with a neutralizing antibody, which highlights the 3-fold region as an important antigenic determinant as well (90). Residues on the 3-fold protrusions of AAV8 also determine receptor binding and transduction efficiency (19,90,91). Cleavage sites near the 5-fold axis did not show up in our maps, indicating that this region is not highly dynamic under the conditions use this region is dynamic under certain conditions, because conformational diversity is present in structural models (1, 3).…”

Structural Insights into Adeno-Associated Virus Serotype 5

“…The surface expo- sure of these residues was also predicted based on a model by Arbetman et al (13). The surface-localized residues occupy analogous regions to those mapped as antigenic footprints on other AAV capsids, for example, AAV2 and AAV8 (97,100,101,114), and thus likely dictate the antigenic differences reported between the two viruses when their transduction efficiencies were compared in the presence of intravenous immunoglobulin (13). Internal capsid volumes are comparable between AAV2, AAV4, and AAV5 and do not support an increased packaging capacity for AAV5 compared to other AAVs.…”

“…The AAV capsid surface features that are unique to AAV5-shorter 3-fold protrusions, extended VR-VII, and smaller HI loop-contain amino acid residues or are proximate to capsid regions reported to play essential roles in the AAV life cycle. For example, residues within VR-IV, VR-V, and VR-VIII which make up the 3-fold protrusions have been reported to control glycan receptor attachment (VR-V and VR-VIII for AAV2 and VR-V for AAV9) (90)(91)(92)(93)122), transduction (VR-IV, VR-V, and VR-VIII in AAV2, VR-VIII in AAV8, and VR-IV, VR-V, and VR-VIII in AAV9) (94)(95)(96)(97)(98)(99) and antigenic phenotypes (VR-IV, VR-V, and VR-VIII in AAV2 and VR-VIII in AAV8 (97,100,101). These regions have similar roles in AAV5.…”