Epitope mapping of human anti-adeno-associated virus type 2 neutralizing antibodies: implications for gene therapy and virus structure

Abstract

Recombinant adeno-associated virus type 2 (AAV) is a common vector used in human gene therapy protocols. We characterized the humoral immune response to AAV and observed that 80% of normal human subjects have anti-AAV antibodies and that 18% have neutralizing antibodies. To analyze the effect of neutralizing antibodies on AAV readministration, we attempted to deliver recombinant AAV expressing human factor IX (AAV-hFIX) intraportally into the livers of mice which had been preexposed to AAV and shown to harbor a neutralizing antibody response. While all naive control mice expressed hFIX following administration of AAV-hFIX, none of the mice with preexisting immunity expressed hFIX, even after transient immunosuppression at the time of the second administration with anti-CD4 or anti-CD40L antibodies. This suggests that preexisting immunity to AAV, as measured by a neutralizing antibody response, may limit AAV-mediated gene delivery. Using human sera in an enzyme-linked immunosorbent assay for AAV and a capsid peptide scan library to block antibody binding, we mapped seven regions of the AAV capsid containing immunogenic epitopes. Using pools of these peptides to inhibit the binding of neutralizing antibodies, we have identified a subset of six peptides which potentially reconstitute a single neutralizing epitope. This information may allow the design of reverse genetic approaches to circumvent the preexisting immunity that can be encountered in some individuals.

FIG. 1

Characterization of the anti-AAV immune response in humans. (A) Representative ELISA using 30 (of the total of 50 tested) serum samples to probe AAV particles. ●, test samples. Controls, including an anti-AAV monoclonal antibody (■) and anti-AAV serum raised in guinea pigs (⧫), verified the specificity of detection in this assay. The dashed line represents the background cutoff. (B) Representative neutralization assay analysis. AAV-GFP was used to infect 293 cells in the presence of human serum. Plot a (shaded), normal GFP expression in the absence of serum; plot b, example of a neutralizing serum sample that blocks AAV uptake and prevents GFP expression; plot c, example of a serum that does not inhibit AAV-GFP uptake into the cells.

FIG. 2

AAV vector delivery into mice with preexisting immunity. (A) Analyses of neutralization antibodies from murine sera collected 42 days after AAV-LacZ administration. Neutralization assays were performed in parallel with serum from each mouse, a positive human serum sample (Ser3), and an AAV-neutralizing monoclonal antibody (A20) at a dilution of 1:500. (B) Serum hFIX levels were measured for 62 days after introduction of AAV-hFIX intraportally into 20 preexposed and 5 naive mice. All 20 mice pretreated with AAV-LacZ (●) showed no hFIX expression over the entire 62 days of follow-up testing. The five naive mice (■, , , , and ) expressed levels varying from 50 to 250 ng of hFIX per ml.

FIG. 3

Mapping of anti-AAV antibody epitopes by ELISA and neutralization assay blocking analyses. (A) Representative analysis of peptide blocking of antibody binding in ELISA using Ser3 and peptides 53 to 62. Positive blocking was considered to cause an inhibition of at least 50% relative to a no-peptide control (no pept). (B) Pools of peptides block neutralizing antibodies. AAV antibody neutralizing assays were performed under the following conditions: plot a, AAV-GFP only; plot b, AAV-GFP with 14-peptide pool; plot c (shaded), AAV-GFP plus neutralizing Ser24; plot d, AAV-GFP plus neutralizing Ser24 in the presence of 14-peptide pool. (C) Plot a, AAV-GFP; plot b, AAV-GFP plus neutralizing Ser24 in the presence of peptide pool 2; plot c, AAV-GFP with neutralizing Ser24; plot d, (shaded), AAV-GFP plus neutralizing Ser24 in the presence of peptide pool 1; plot e, AAV-GFP plus neutralizing Ser24 in the presence of negative peptide pool.

FIG. 4

Summary of antibody epitope mapping. Each box represents a 15-amino-acid peptide sequence from AAV VP1 starting at MAADGY and ending with LTRNL. A total of 91 peptides overlapping by five amino acids were used. The VP2 sequence begins with TAPGK (amino acid 149, peptide 17), and the VP3 sequence begins with MATGS (amino acid 203, peptide 25). Blackened boxes represent a blocking of antibody binding by this peptide in ELISA. Blocking peptide numbers are shown for reference above and below the grid. Serum sample designations are shown for reference to the left of the grid. Asterisks mark those sera that were positive for neutralizing antibodies.

FIG. 5

Sequences of immunogenic peptides identified by peptide blocking ELISA experiments. Overlapping sequences from two positive peptides are underlined and shown as putative epitopes, and overlapping sequences from three juxtaposed peptides are double underlined. The shaded area corresponds to the conformational epitope sequences.

FIG. 6

Structural locations of the immunogenic regions of AAV. (A) Amino acid sequences of the overlapping VP1, VP2, and VP3 proteins that form the AAV capsid. The arrows indicate the start point of the protein sequences of VP1, VP2, and VP3. Identified epitopes are underlined in bold and marked with the corresponding peptide designation; “lip” denotes the insertion site of four amino acids that result in lip mutants (7). The basic regions proposed to interact with heparan sulfate proteoglycan are marked with a checkered line. The structural regions extrapolated from the CPV structure are marked above the corresponding sequence. ▴, key residues involved in determining tropism of CPV; dashed box, the VFTDSE sequence recognized by CPV-neutralizing dog serum. (B) Schematic representation of parvovirus structure (adapted from reference 12) that shows the approximate structural locations of the epitopes identified in this study. The icosohedral structure (left) is composed of 60 icosohedral units (shaded triangle) formed by VP1, VP2, and VP3. The expanded triangle represents one icosohedral unit.