An investigation of the immune epitope properties of adeno-associated virus capsid-derived peptides among hemophilia patients

Adeno associated virus (AAV) is an optimal gene vector for monogenic disorders. However, neutralizing antibodies (Nab) against AAV hinder its widespread application in gene therapy. In this study, we biosynthesized peptides recognized by the Binding Antibodies (Bab) from the sera containing high Nab titer against AAV2. We established four immunological methods to detect immune epitopes of the AAV2-derived peptides, including Bab assay, Nab assay, B-cell receptors (BCR) detecting assay and immunoglobin-producing B cells enzyme-linked immunosorbent spot (B-cell ELISpot) assay. Correlations among the epitopes determined by these four methods were analyzed using the serum samples and peripheral blood mononuclear cells (PBMC) from 89 hemophilia A/B patients. As decoys, the peptides' ability to block the Nab of AAV2 particles was assessed using AAV transduction models both in vitro and in vivo. Overall, we provide insights into AAV2 capsid-derived peptides immune epitopes, involving the Nab, Bab, BCR, and B-cell ELISpot, offering alternative immunological evaluation approaches and strategy to overcome Nab barriers in AAV-mediated gene therapy.