Adeno-Associated Virus Serotype 1 (AAV1)- and AAV5-Antibody Complex Structures Reveal Evolutionary Commonalities in Parvovirus Antigenic Reactivity

Abstract

The clinical utility of the adeno-associated virus (AAV) gene delivery system has been validated by the regulatory approval of an AAV serotype 1 (AAV1) vector for the treatment of lipoprotein lipase deficiency. However, neutralization from preexisting antibodies is detrimental to AAV transduction efficiency. Hence, mapping of AAV antigenic sites and engineering of neutralization-escaping vectors are important for improving clinical efficacy. We report the structures of four AAV-monoclonal antibody fragment complexes, AAV1-ADK1a, AAV1-ADK1b, AAV5-ADK5a, and AAV5-ADK5b, determined by cryo-electron microscopy and image reconstruction to a resolution of ϳ11 to 12 Å. Pseudoatomic modeling mapped the ADK1a epitope to the protrusions surrounding the icosahedral 3-fold axis and the ADK1b and ADK5a epitopes, which overlap, to the wall between depressions at the 2-and 5-fold axes (2/5-fold wall), and the ADK5b epitope spans both the 5-fold axis-facing wall of the 3-fold protrusion and portions of the 2/5-fold wall of the capsid. Combined with the six antigenic sites previously elucidated for different AAV serotypes through structural approaches, including AAV1 and AAV5, this study identified two common AAV epitopes: one on the 3-fold protrusions and one on the 2/5-fold wall. These epitopes coincide with regions with the highest sequence and structure diversity between AAV serotypes and correspond to regions determining receptor recognition and transduction phenotypes. Significantly, these locations overlap the two dominant epitopes reported for autonomous parvoviruses. Thus, rather than the amino acid sequence alone, the antigenic sites of parvoviruses appear to be dictated by structural features evolved to enable specific infectious functions. IMPORTANCEThe adeno-associated viruses (AAVs) are promising vectors for in vivo therapeutic gene delivery, with more than 20 years of intense research now realized in a number of successful human clinical trials that report therapeutic efficacy. However, a large percentage of the population has preexisting AAV capsid antibodies and therefore must be excluded from clinical trials or vector readministration. This report represents our continuing efforts to understand the antigenic structure of the AAVs, specifically, to obtain a picture of "polyclonal" reactivity as is the situation in humans. It describes the structures of four AAV-antibody complexes determined by cryoelectron microscopy and image reconstruction, increasing the number of mapped epitopes to four and three, respectively, for AAV1 and AAV5, two vectors currently in clinical trials. The results presented provide information essential for generating antigenic escape vectors to overcome a critical challenge remaining in the optimization of this highly promising vector delivery system. T he adeno-associated viruses (AAVs), single-stranded DNA packaging viruses belonging to the Parvoviridae family, are promising vectors for gene delivery. There are over 100 AAV genomic isolates, and 13 human and nonhuman serotypes h...

Characterization of the Adeno-Associated Virus 1 and 6 Sialic Acid Binding Site

“…Mutagenesis combined with cell binding and transduction assays confirmed N447, S472, V473, N500, T502, and W503 as being important for AAV1 and AAV6 SIA interaction. In addition, an overlap between the SIA binding site and the AAV1 ADK1a antigenic epitope (41) was confirmed by native dot blot analysis, enzyme-linked immunosorbent assay (ELISA), and antibody neutralization assay, suggesting steric hindrance of receptor attachment as the neutralization mechanism of this antibody. This study thus provides information for AAV1 and AAV6 determinants of cellular recognition, as well as for their engineering to escape from antibody recognition.…”

“…To overcome this challenge, capsid modification based on capsid antigenic epitopes can generate variants with the ability to escape from antibody recognition (25,72). The antigenic structures of AAV1, AAV2, AAV5, AAV6, and AAV8 have been characterized by cryo-electron microscopy and image reconstruction (41,58,73,74). Despite sequence diversity (ϳ60 to 99%) between these serotypes, two common antigenic regions were identified which are located on the 2/5-fold wall and 3-fold protrusion on the capsid surface (41).…”

“…The antigenic structures of AAV1, AAV2, AAV5, AAV6, and AAV8 have been characterized by cryo-electron microscopy and image reconstruction (41,58,73,74). Despite sequence diversity (ϳ60 to 99%) between these serotypes, two common antigenic regions were identified which are located on the 2/5-fold wall and 3-fold protrusion on the capsid surface (41). Although their mechanisms of neutralization are mostly unknown, some of these antigenic footprints coincided with regions determining receptor binding and regions controlling transduction phenotype.…”

“…10600007) using the Spot-Blot system (GE Healthcare). The unboiled vectors were blotted with hybridoma supernatant for ADK1b and 5H7 and purified antibodies for ADK1a and 4E4 produced as previously described (41,58). Briefly, hybridoma supernatant isolated from cells producing the MAb was purified by Hi-Trap protein G HP columns (GE Healthcare).…”

Cryo-electron Microscopy Reconstruction and Stability Studies of the Wild Type and the R432A Variant of Adeno-associated Virus Type 2 Reveal that Capsid Structural Stability Is a Major Factor in Genome Packaging

“…The tops of these insertion loops are classified into nine major variable regions (VRs) that vary in sequence and structure among the different serotypes. These VRs are associated with specific functional roles, including receptor attachment, transduction phenotype, and antigenicity, for each of the AAV serotypes (15,(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34). The VPs assemble a capsid whose morphology is characterized by surface depressions at the icosahedral 2-fold axes of symmetry, three protrusions surrounding a depression at each 3-fold axis, and a pore at each 5-fold axis surrounded by a shallow (canyon-like) depression (14-23).…”