FIG 4.

Analysis of AAV9 antibody escape variants. (A) SDS-PAGE and negative stain EM of WT AAV9 and variants. (B to E) Native dot immunoblots of AAV9 variants against AAV9 antibodies and transduction analysis compared to WT AAV9. Substitutions in variants of (B) VR-IV LS and (C) S454A are localized to the 3-fold axis of symmetry and exhibit escape from HL2370 and HL2374, while (D) P659K includes a substitution in the HI-loop and escapes HL2372. Panel E shows a combination S454A/P659K and escapes the three MAbs, HL2370, HL2372, and HL2374. The absence of a black dot in the native dot blots indicates loss of recognition of that variant by the antibody. WT AAV9 serves as a positive control and is recognized by all the cognate antibodies, while WT AAV5 serves as a negative control and is only recognized by its cognate antibody, ADK5b. ADK9 is as a positive native capsid loading control. B1 is a loading control and recognizes the C-terminal residues of denatured viral proteins (VP1, VP2, and VP3).