You are using an outdated browser. This page doesn’t support Internet Explorer 6, 7 and 8.Please upgrade your browser or activate Google Chrome Frame to improve your experience. The recombinant adeno-associated virus (rAAV) gene delivery system is entering a crucial and exciting phase with the promise of more than 20 years of intense research now realized in a number of successful human clinical trials. However, as a natural host to AAV infection, anti-AAV antibodies are prevalent in the human population. For example, ~70% of human sera samples are positive for AAV serotype 2 (AAV2). Furthermore, low levels of pre-existing neutralizing antibodies in the circulation are detrimental to the efficacy of corrective therapeutic AAV gene delivery. A key component to overcoming this obstacle is the identification of regions of the AAV capsid that participate in interactions with host immunity, especially neutralizing antibodies, to be modified for neutralization escape. Three main approaches have been utilized to map antigenic epitopes on AAV capsids. The first is directed evolution in which AAV variants are selected in the presence of monoclonal antibodies (MAbs) or pooled human sera. This results in AAV variants with mutations on important neutralizing epitopes. The second is epitope searching, achieved by peptide scanning, peptide insertion, or site-directed mutagenesis. The third, a structure biology-based approach, utilizes cryo-electron microscopy and image reconstruction of AAV capsids complexed to fragment antibodies, which are generated from MAbs, to directly visualize the epitopes. In this review, the contribution of these three approaches to the current knowledge of AAV epitopes and success in their use to create second generation vectors will be discussed. Figure 1. The AAV capsid. Radially color-cued (from capsid center to surface: blue-green-yellow-red, ~110–130 Å) of the AAV1 capsid generated from 60 VP monomers (RCSB PDB # 3NG9). The approximate icosahedral 2-, 3-, and 5-fold symmetry axes are as well as the AAV capsid surface features are indicated by the arrows and labeled. This image was generated using the Chimera program (40). Figure 2. AAV variable regions. (A) A ribbon diagram representation of the ordered overlapping VP3 monomer region of AAV1. The conserved β-barrel core motif (βBIDG-βCHEF, gray), conserved αA helix, DE loop (between βD and βE), HI loop (between βH and βI), VR-I to VR-IX [defined (26)] are colored, I: purple, II: blue, III: yellow, IV: red, V: black, VI: hot pink, VII: cyan, VIII: green, and IX: brown, and labeled. The approximate positions of the 2-, 3-, and 5-fold axes are indicated by the filled oval, triangle, and pentagon, respectively. The N and C labels are the N- and C-terminal ends of the ordered VP region, respectively. (B) The capsid surface of AAV2 with VR-I to VR-IX colored as in (A). The approximate icosahedral 2-, 3-, and 5-fold symmetry axes are indicated and labeled as in Figure 1. Both (A) and (B) were generated with the PyMOL program (http://www.pymol.org). Figure 3. Structurally mapped AAV antigenic epitopes. The epitopes identified on the AAV capsid surface by cryo-reconstruction structure are depicted in the colors used for the VRs in Figure 2 based on overlap with the VR amino acids, aa253–271: purple, aa383–386: yellow, aa456–459: red, aa492–515: black, aa544–557: cyan, aa582–597: green, aa659–669: wheat, and aa709–720: brown. Amino acids 659–669 (wheat) were not previously described as VR regions. The approximate icosahedral 2-, 3-, and 5-fold symmetry axes are indicated and labeled as in Figure 1. This image was generated with the PyMOL program (http://www.pymol.org). Citation: Tseng Y-S and Agbandje-McKenna M (2014) Mapping the AAV capsid host antibody response toward the development of second generation gene delivery vectors. Front. Immunol. 5:9. doi: 10.3389/fimmu.2014.00009 Copyright: © 2014 Tseng and Agbandje-McKenna. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. *Correspondence: Mavis Agbandje-McKenna, Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville, FL 32610, USA e-mail: firstname.lastname@example.org Source.