Journal: Viruses

Article Title: The Structure of an AAV5-AAVR Complex at 2.5 Å Resolution: Implications for Cellular Entry and Immune Neutralization of AAV Gene Therapy Vectors

doi: 10.3390/v12111326

Figure Lengend Snippet: Overlap in the binding sites on AAV5 of receptor AAV5 and antibodies ADK5a & ADK5b. ( A ) The solvent accessible surface of AAV5 is colored with the ADK5a footprint blue, AKD5b red, and amino acids common to both, purple. The footprints comprise residues identified in prior EM structures at 11 Å resolution . Three-fold symmetry axes, marked as triangles, map to equatorial three-folds left and right of center in B, while the five-fold (pentagon) maps to a five-fold above center on the mid-line of B. The view in in B and here is down a two-fold axis. ( B ) The current structure of AAVR’s PKD1 is overlaid (with its symmetry equivalents), showing that the antibody footprints are substantially occluded. ( C – E ) Roadmap projections of the AAV5 surface colored by distance from the center of the virus. The perspective is the same as in panels A & B. The triangle shows one unique asymmetric unit of the surface, which, if expanded 60-fold by the icosahedral symmetry, would generate the entire virus surface. ( C ) The contact footprint of AAVR PKD1 is outlined in white. It is concentrated on the side of a three-fold spike, and although the domain occludes access to a swath leading up towards the five-fold, there is not the previously reported contact near the five-fold pore. A magnified version is provided in the supplement with residues labeled. ( D,E ) illustrate the footprints of ADK5b and ADK5a superimposed on that of AAVR PKD1, showing substantial overlap.

Article Snippet: To test the competition of various concentrations of PKD1 with a constant concentration of the AAV5 antibodies ADK5a (Progen, Heidelberg, Germany, cat. no. 615148) or ADK5b (Origene, Rockville, MD, USA, cat. no. AM09121PU-N), PKD1 was diluted in buffer (25 mM HEPES, 125 mM sodium chloride at pH 7.4) containing roughly 2 nM of ADK5a or 1 nM of ADK5b to a PKD1 concentration of 6 μM.

Techniques: Binding Assay, Solvent, Virus, Data-independent acquisition, Labeling

Journal: Viruses

Article Title: The Structure of an AAV5-AAVR Complex at 2.5 Å Resolution: Implications for Cellular Entry and Immune Neutralization of AAV Gene Therapy Vectors

doi: 10.3390/v12111326

Figure Lengend Snippet: Competition assays of ADK5a, ADK5b, and PKD1. ( A ) ELISA of antibody binding to AAV5 with constant antibody concentration and differing levels of PKD1 concentration, performed as six replicates and normalized to a positive control without PKD1. ( B ) ELISA of PKD1 binding to AAV5 with a constant PKD1 concentration of 0.1µM and various levels of ADK5a performed in triplicate and normalized to a positive control without antibody. ( C ) The same as B but with ADK5b instead of ADK5a. All error bars are ± 1 standard deviation.

Article Snippet: To test the competition of various concentrations of PKD1 with a constant concentration of the AAV5 antibodies ADK5a (Progen, Heidelberg, Germany, cat. no. 615148) or ADK5b (Origene, Rockville, MD, USA, cat. no. AM09121PU-N), PKD1 was diluted in buffer (25 mM HEPES, 125 mM sodium chloride at pH 7.4) containing roughly 2 nM of ADK5a or 1 nM of ADK5b to a PKD1 concentration of 6 μM.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Concentration Assay, Positive Control, Standard Deviation