Prevalence study of cellular capsid-specific immune responses to AAV2, 4, 5, 8, 9 and rh10 in healthy donors

Recombinant adeno-associated virus (rAAV) vectors appear, more than ever, to be efficient viral vectors for in vivo gene transfer as illustrated by the approvals of 7 drugs across Europe and the USA. Nevertheless, pre-existing immunity to AAV capsid in humans remains one of the major limits for a successful clinical translation. Whereas pre-existing humoral response to AAV capsid is well documented, the prevalence of pre-existing capsid-specific T cell responses still needs to be studied and characterized. Here, we investigated the prevalence of AAV-specific circulating T cells towards AAV2, 4, 5, 8, 9 and rh10 in a large cohort of healthy donors using the standard IFNγ ELISpot assay. We observed the highest prevalence of pre-existing cellular immunity to AAV9 serotype followed by AAV8, AAV4, AAV2, AAVrh10 and AAV5 independently of the donors’ serological status. An in-depth analysis of T cell responses towards the 2 most prevalent serotypes 8 and 9 shows that IFNγ secretion is mainly mediated by CD8 T cells for both serotypes. A polyfunctional analysis reveals different cytokine profiles between AAV8 and AAV9. Surprisingly, no IL-2 secretion was mediated by anti-AAV9 immune cells suggesting that these cells may rather be exhausted or terminally differentiated than cytotoxic T cells. Altogether, these results suggest that pre-existing immunity to AAV may vary depending on the serotype and support the necessity of using multiparametric monitoring methods to better characterize anti-capsid cellular immunity and foresee its impact in rAAV-mediated clinical trials. ### Competing Interest Statement A.A., M.D., N.J., C.C., J.L.D., A.C., M.G., O.A. and G.G. declare no competing financial interests with regards to this work. R.X. is an employee of Roche, Basel, Switzerland. C.V. is an employee of Charles River Laboratories, Evreux, France