Author

Dustin McCraw

Date

May 2012

Document Type

Dissertation

Degree Name

Ph.D.

Department

Dept. of Biochemistry and Molecular Biology

Institution

Oregon Health & Science University

Abstract

Adeno--associated virus (AAV) is a leading candidate as a gene therapy vector, but as with other vectors, one of the limitations is a host neutralizing immune response. The most extensively characterized immune interaction is that between AAV serotype 2 and neutralizing monoclonal antibody (MAb) A20. The monovalent A20 Fab fragment and AAV--2 were initially isolated for structural studies. A20 monoclonal antibody is confirmed to have high binding affinity to AAV--2, however A20 Fab is shown to have only low binding affinity and a complex could not be isolated. Alternative production methods show that A20 Fab’ exhibits a relatively high binding affinity to AAV--2. A cryo--electron microscopy (EM) structure of AAV--2 complexed with the Fab’ fragment of A20 has been determined to 8.5 Å resolution. The binding footprint is determined through fitting the cryo--EM reconstruction with a homology model following sequencing of the variable domain, and provides a structural basis for integrating diverse prior epitope mappings. Direct visualization sheds new light on prior experiments, resolving inconsistencies in proposed components of the epitope. The A20 footprint on AAV--2 extends from the plateau, a region of moderate sequence diversity implicated in some of the prior studies, to the side of the spike, and into the conserved canyon, covering a much larger area than anticipated. Comparison with structures of binding and non--binding serotypes indicates that recognition depends on a combination of subtle serotype--specific features. Separation of the neutralizing epitope from the heparan sulfate cell attachment site encourages attempts to develop immune--resistant vectors that can still bind to target cells. Additional assessment of the electrostatic surface and the free energy of solvation of the binding site show that there is no singular distinguishing characteristic between AAV serotypes that bind to A20 and those which do not. The current structure suggests that A20 recognition is therefore moderated by several relatively small modulations in serotype differences at the binding site.

Identifier

doi:10.6083/M4154F2N

School

School of Medicine

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