title: Mapping a neutralizing epitope onto the capsid of adeno-associated virus serotype 8.
abstract: 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida, USA.
authors.forename: Brittney L
authors.initials: BL
authors.lastname: Gurda
authors.forename: Christina
authors.initials: C
authors.lastname: Raupp
authors.forename: Ruth
authors.initials: R
authors.lastname: Popa-Wagner
authors.forename: Matthias
authors.initials: M
authors.lastname: Naumer
authors.forename: Norman H
authors.initials: NH
authors.lastname: Olson
authors.forename: Robert
authors.initials: R
authors.lastname: Ng
authors.forename: Robert
authors.initials: R
authors.lastname: McKenna
authors.forename: Timothy S
authors.initials: TS
authors.lastname: Baker
authors.forename: Jürgen A
authors.initials: JA
authors.lastname: Kleinschmidt
authors.forename: Mavis
authors.initials: M
authors.lastname: Agbandje-McKenna
mesh_terms: D021581:Active Transport, Cell Nucleus; D000818:Animals; D000914:Antibodies, Viral / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D000956:Antigens, Viral / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D036022:Capsid Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D002467:Cell Nucleus / Q000235:genetics / Q000276:immunology / Q000821:virology; D018360:Crystallography, X-Ray; D000229:Dependovirus / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D018604:Epitope Mapping; D005260:Female; D018014:Gene Transfer Techniques; D057809:HEK293 Cells; D006367:HeLa Cells; D056945:Hep G2 Cells; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D051379:Mice; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship
chemical_list: D000914:Antibodies, Viral; D000956:Antigens, Viral; D036022:Capsid Proteins; D007140:Immunoglobulin Fab Fragments