697. Phenotypic and Functional Characterisation of Human Anti-AAV CD8+ T Cells Using MHC Class I Tetramer-Associated Magnetic Enrichment

Abstract

PBS-formulated rAAV2/8 PGK GFP-HY were injected subretinally in C57Bl/6 female mice. At day 7, mice were challenged subcutaneously with the UTY and DBY peptides adjuvanted in CFA, and the immune response was analyzed at day 14 by IFNγ ELISpot, cytokine titration and proliferation assays. Our results revealed that: (i) The subretinal injection of 10E8 to 2.10E9 vg/mouse of rAAV2/8 PGK GFP-HY did not induce a significant HY-specific peripheral immune modulation in contrast to the ACAID obtained after subretinal injection of UTY and DBY peptides (50µg each); (ii) Higher doses of rAAV2/8 PGK GFP-HY (5.10E10 vg/mouse) triggered increased Th1 and Tc1 cellular immune responses against the transgene product in peripheral lymphoid organs. Lower doses of vector did not increase peripheral immune responses toward the transgene product following the challenge. We have thus far failed to demonstrate the induction of ACAID by rAAV2/8 subretinal gene transfer. We show that rAAV2/8 vector-mediated subretinal gene transfer is not necessarily ignored at the immunological level. High doses of vector can effectively trigger anti-transgene T-cell responses with the potential for elimination of transgene-expressing cells. Clearly, anti-transgene-specific immune monitoring should be refined at least in preclinical models, to improve the biosafety and the long-term efficacy of rAAV-mediated ocular gene transfer.