Mutations on the external surfaces of adeno-associated virus type 2 capsids that affect transduction and neutralization.
Mutations were made at 64 positions on the external surface of the adeno-associated virus type 2 (AAV-2) capsid in regions expected to bind antibodies. The 127 mutations included 57 single alanine substitutions, 41 single nonalanine substitutions, 27 multiple mutations, and 2 insertions. Mutants were assayed for capsid synthesis, heparin binding, in vitro transduction, and binding and neutralization by murine monoclonal and human polyclonal antibodies. All mutants made capsid proteins within a level about 20-fold of that made by the wild type. All but seven mutants bound heparin as well as the wild type. Forty-two mutants transduced human cells at least as well as the wild type, and 10 mutants increased transducing activity up to ninefold more than the wild type. Eighteen adjacent alanine substitutions diminished transduction from 10- to 100,000-fold but had no effect on heparin binding and define an area (dead zone) required for transduction that is distinct from the previously characterized heparin receptor binding site. Mutations that reduced binding and neutralization by a murine monoclonal antibody (A20) were localized, while mutations that reduced neutralization by individual human sera or by pooled human, intravenous immunoglobulin G (IVIG) were dispersed over a larger area. Mutations that reduced binding by A20 also reduced neutralization. However, a mutation that reduced the binding of IVIG by 90% did not reduce neutralization, and mutations that reduced neutralization by IVIG did not reduce its binding. Combinations of mutations did not significantly increase transduction or resistance to neutralization by IVIG. These mutations define areas on the surface of the AAV-2 capsid that are important determinants of transduction and antibody neutralization.
Lochrie
Michael A
MA
Avigen, Inc., Alameda, California 94502-6541, USA.
Tatsuno
Gwen P
GP
Christie
Brian
B
McDonnell
Jennifer Wellman
JW
Zhou
Shangzhen
S
Surosky
Richard
R
Pierce
Glenn F
GF
Colosi
Peter
P
D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000914:Antibodies, Viral / Q000276:immunology*; D002213:Capsid / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D000229:Dependovirus / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D006493:Heparin / Q000378:metabolism; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D051379:Mice; D008958:Models, Molecular; D009154:Mutation; D009500:Neutralization Tests; D014161:Transduction, Genetic
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D007106:Immune Sera; D006493:Heparin; D000409:Alanine