Identification of the murine AAVrh32.33 capsid-specific CD8+ T cell epitopes.

Abstract

Adeno-associated virus (AAV) is an ideal gene therapy vector and is non-immunogenic in many small animal models. The stable gene expression commonly seen in murine models does not necessarily translate to nonhuman primates and higher-order species, highlighting the need for a better understanding of immune activation to these vectors. One capsid variant, AAVrh32.33, demonstrates a unique phenotype in murine muscle, reminiscent of what is often seen in higher-order species. AAVrh32.33 generates a strong CD8+ T-cell response to both capsid and encoded transgene antigens in a manner independent of transgene product or major histocompatability complex haplotype, making it an ideal candidate for studying immune activation to AAV in the mouse. To map the H-2b and H-2d dominant epitopes of the AAVrh32.33 capsid, C57BL/6 or Balb/C mice received an intramuscular injection of 1 x 10(11) genome copies of AAV2/rh32.33.CB.nLacZ. Three weeks later, splenocytes were harvested and stimulated in vitro with pooled or individual peptides from the AAVrh32.33 capsid peptide library and analysed by an interferon (IFN)-gamma enzyme-linked immunosorbent spot assay or intracellular cytokine staining. The immunodominant epitopes within the AAVrh32.33 capsid responsible for driving CD8+ T-cell responses to the capsid protein in C57BL/6 (SSYELPYVM) and Balb/C (KIPASGGNAL) mice were defined. Identification of dominant capsid epitopes will make it possible to monitor cellular responses to the AAV capsid in vivo, facilitating mechanistic studies critical to defining how cellular immunity to the AAV capsid arises and, ultimately, how the generation of capsid-specific T cells can be avoided to ensure safety in a gene therapy setting.