Structural and antigenic characterization of the avian adeno-associated virus capsid

Abstract

AAVs are extensively studied as promising therapeutic gene delivery vectors. In order to circumvent pre-existing antibodies targeting primate-based AAV capsids, the AAAV capsid was evaluated as an alternative to primate-based therapeutic vectors. Despite the high sequence diversity, the AAAV capsid was found to bind to a common glycan receptor, terminal galactose, which is also utilized by other AAVs already being utilized in gene therapy trials. However, contrary to the initial hypothesis, AAAV was recognized by approximately 30% of human sera tested. Structural and sequence comparisons point to conserved epitopes in the fivefold region of the capsid as the reason determinant for the observed cross-reactivity.

Keywords: AAV vector; adeno-associated virus; antigenicity; avian viruses; capsid; cryo-EM; gene therapy; parvovirus; three-dimensional structure.

Fig 1

Production and purification of AAAV viral capsids. (A) A two-dimensional dendrogram is shown, constructed using the hierarchical clustering analysis platform Phylogeny.fr (40) and the VP1 amino acid sequences of selected dependoparvoviruses. (B) SDS gel of purified AAAV. AAV5 was loaded as a control. The position of VP1, VP2, and VP3 is indicated. The VPs of AAV5 are known to migrate higher than their actual molecular weight (41), despite being 1–3 kDa lower compared to AAAV. (C) Cryo-EM micrograph with full (black arrow) and empty (white arrow) AAAV capsids. Scale bar: 500 Å.

Fig 2

Genome-containing and empty AAAV capsid structures. (A) Capsid surface density maps determined by cryo-EM reconstruction contoured at a sigma (σ) threshold level of 1. The resolutions of the structures are calculated based on an FSC threshold of 0.143. The reconstructed maps are radially colored (blue to red) according to distance to the particle center, as indicated by the scale bar below. The icosahedral two, three, and fivefold symmetry axes are indicated on the full AAAV capsid map. (B) Cross-sectional views of the reconstructed maps from genome-containing and empty particles contoured at a σ-threshold level of 0.9. The positions of some icosahedral two-, three-, and fivefold symmetry axes are indicated by arrowheads. This figure was generated using UCSF-Chimera (48).

Fig 3

Differences between the density maps of the empty and full AAAV capsid structures. (A) Modeled AAAV resides at the fivefold/N-terminus of AAAV (B) in VR-IX, and (C) at the DNA binding pocket (red = full, black = empty).

Fig 4

AAAV VP monomer and capsid arrangement. (A) Structural superposition of AAAV full (pink) and empty (light pink) monomers is shown as ribbon diagrams with the position of VR-I to VR-IX, the HI-loop, β-strand A-I, α-helix A, the N- and C-terminus, and the icosahedral two-, three-, and fivefold axes are labeled. This image was generated using PyMOL (60). (B) Four AAAV VP monomers A–D arranged to form the intersection of the icosahedral symmetry axes at the two-, three-, and fivefold wall.

Fig 5

Structural comparison of AAAV monomeric viral protein against AAV serotypes 2, 5, and 9. (A) Table of Cα distances and amino acid sequence identity of major VP of AAAV compared to AAV2, AAV5, and AAV9. (B) Structural superposition of AAAV (pink), AAV2 (blue), AAV5 (gray), and AAV9 (brown) shown as ribbon diagrams. The position of the N- and C-terminus, and the icosahedral two-, three-, and fivefold axes are labeled. This figure was generated using PyMol (60).

Fig 6

AAAV binds to the terminal galactose glycan receptor. (A) Luciferase-packing AAV viral capsids were incubated with CHO Pro5 and Lec2 cells as described in the Materials and Methods section. The cell-binding efficiency was determined via a qPCR-based assay, detecting the luciferase gene. The results are normalized to CHO Pro5. AAV2 was tested as a positive control for all cell lines as it binds to heparan sulfate proteoglycan. AAV5 was included as a sialic acid binding control. AAV9 was included as a galactose binding control. (B) AAAV, AAV9, and AAVrh.10 all bind to the galactose glycan receptor. Comparison of the key residues involved in the galactose binding pocket for the three types of AAVs.

Fig 7

AAAV is neutralized by ~30% of human serum samples from 50 healthy individuals. (A) Neutralization assays conducted in DF-1 cells for AAAV. For AAV2, AAV5, and AAV9 neutralization assays were conducted in HEK293 cells. Cells were incubated with purified virus at an MOI = 10⁵ in human serum samples. Normalized transduction units of AAAV, AAV2, AAV5, and AAV9 were determined by a luciferase gene reporter assay. All experiments were carried out in triplicate (n = 3). (B) Native dot immunoblots of AAAV, AAV9, and AAV5 against mAb2-7 (56) and B1 with 10¹⁰ loaded capsid particles/dot. (C) Low resolution structure of an AAAV capsid complexed with Fab2-7 determined using cryo-EM to 13 Å resolution. (D) Surface representation of the AAAV capsid highlighting the fivefold region and amino acid sequence alignments comparing the BC-loop (purple), DE-loop (blue), and HI-loop (orange) between AAV2, AAV5, AAV9, and AAAV.