Generation of cynomolgus monkey capsid-specific positive control cells for IFNγ ELISpot assays for adeno-associated gene therapy applications.

Cell-mediated immune (CMI) responses to adeno-associated virus (AAV) can lead to tissue damage and loss of therapeutic transgene expression. Identifying robust biomarkers and mechanisms of CMI can aid clinical practice and advancement of AAV gene therapies. The present work evaluated peripheral blood mononuclear cells (PBMC) from non-human primates (NHP) before and after immunization with adenovirus 5 encoding AAV9 capsid antigen. PBMC were stimulated ex vivo with AAV9 capsid peptides to evaluate CMI responses by interferon (IFN)-γ ELISpot, intracellular cytokines/activation markers, secreted cytokines, and RNAseq. AAV peptide stimulation produced a robust IFNγ ELISpot 11 days after immunization and ≈ 4 years after cryopreservation. Flow cytometry revealed increased IFNγ, interleukin (IL)-2, or tumor necrosis factor (TNF)-positive T-cells. Increases in secreted CXCR3 ligands (IP-10, I-TAC) were detected. Robust changes and correlations to ELISpot responses were revealed by RNAseq, including IFNγ, IP-10, and I-TAC, many downstream transcripts, and several IFN-independent pathways. These data from AAV-immunized NHP identify biomarkers that could serve as robust and sensitive supplements/alternatives to ELISpot for early detection of CMI responses. Assessment of these biomarkers in non-clinical and clinical studies is a critical next step to determine the translation of this work to administration of a therapeutic AAV vector.