4.6 Article

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

Journal

JOURNAL OF VIROLOGY
Volume 86, Issue 15, Pages 7739-7751

Publisher

AMER SOC MICROBIOLOGY
DOI: 10.1128/JVI.00218-12

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Funding

  1. NIH [R21AI072341, R01 GM082946, R37 GM033050, 1S10 RR020016, 2P41RR001081]
  2. University of California, San Diego (UCSD)
  3. Agouron Foundation
  4. Deutsche Forschungsgemeinschaft [KL516/7-1]
  5. National Institute of General Medical Sciences [9P41GM103311]

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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 VPI 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.

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