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 (AAVhFIX) 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.

Neutralizing antibodies against adeno-associated virus examined prospectively in pediatric patients with hemophilia

“…17 Briefly, 293 cells or U87MG cells were seeded in a 48-well plate at a density of 10 5 cells/well in 200 ml DMEM containing 10% FBS. The cells were cultured for 3-4 h at 37 1C and allowed to adhere to the well.…”

“…In approximately 20-60% of this subpopulation, an adaptive humoral response results in the development and maintenance of antibodies capable of neutralizing subsequent infection by AAV2 (neutralizing antibodies (NAbs)). [8][9][10][11][12][13][14][15][16][17][18][19] The existence of NAbs can limit rAAV therapeutic applications that rely on systemic vector administration, for example, by interfering with cell surface binding or promoting reticuloendothelial clearance and destruction of AAV vectors. [20][21][22] Additionally, previous human studies have observed that NAbs against AAV2 cross-neutralize other serotypes of AAV.…”

Library selection and directed evolution approaches to engineering targeted viral vectors

“…Despite these advantageous properties, AAV suffers from several shortcomings, including the inability to target delivery to specific cell types (Muzyczka and Warrington, 2005), inefficient gene delivery to a number of ''nonpermissive'' cell types (Hughes et al, 2002;Ponnazhagan et al, 1996;Smith-Arica et al, 2003;Stacchini et al, 1999), a limited packaging insert size (Dong et al, 1996), and the prevalence of pre-existing immunity to human AAV serotypes in the human population (Moskalenko et al, 2000;Sun et al, 2003).…”

“…Importantly, two rounds of evolution were required for variants to accumulate these two key mutations, highlighting the advantages of multiple rounds of evolution. The former mutation lies in the heparin-binding domain, and the latter has not been associated with a known function, though both have been associated with B cell epitopes (Moskalenko et al, 2000;Opie et al, 2003). Importantly, two novel variants evolved in vitro to evade antibodies mediated efficient delivery in vivo in the presence of neutralizing serum levels 1-3 orders of magnitude higher than those needed to completely neutralize wild-type capsid.…”

“…In light of this high ratio of biological function per nucleotide, when engineering one function, care must therefore be taken to avoid impairing others. Finally, viral properties whose molecular basis is distributed throughout the primary sequence of the capsid, such as virus-cell interactions (Chiorini et al, 1999;Opie et al, 2003) and antibody neutralization (Moskalenko et al, 2000;Wobus et al, 2000), will be extremely challenging to rationally re-design, even with the availability of AAV structures (Xie et al, 2002). Given these numerous complexities and constraints, library selection and directed evolution are particularly well suited for re-engineering viruses.…”