Key result
AAV peptide presentation increases IFNγ and IL-2-producing cells, revealing an immunogenic AAV9 capsid region.
Why the study?
T cells may target vector-transduced and healthy immune cells, impacting safety and efficacy, prompting the need to optimize assays measuring T cell responses against AAV peptide antigens.
Does stimulation with AAV peptide pools elicit a measurable T cell response in PBMCs from healthy donors?
Does stimulation with AAV peptide pools elicit a measurable T cell response in PBMCs from healthy donors?
The optimized ELISpot assay can effectively measure anti-AAV immune responses, providing a tool to assess the immunogenic potential of novel recombinant AAV vectors.
ELISpot assay enables in vitro AAV immunogenicity assessment; leaves open clinical relevance in gene therapy.
) T cells may target vector-transduced cells as well as healthy immune cells, impacting safety and efficacy. In this study, we describe the optimization and reproducibility of interferon-γ (IFNγ)-based and interleukin-2 (IL-2)-based enzyme-linked immunosorbent spot (ELISpot) assays for measuring T cell responses against AAV peptide antigens. For method optimization, peripheral blood mononuclear cells (PBMCs) were isolated from healthy human donors and stimulated with commercially available major histocompatibility complex (MHC) class I or II-specific peptides as positive controls. Peptide pools were designed from published AAV8 and AAV9 capsid protein sequences and then used to assess the presence of AAV-specific T cell responses. Our results demonstrate a measurable increase in IFNγ and IL-2-producing cells after AAV peptide presentation. Furthermore, there was an observed difference in the magnitude and specificity of response to peptide pools based on AAV serotype and donor. Finally, using individual peptides, we identified a region of the AAV9 capsid protein that can elicit an immunogenic response. This work shows the applicability of ELISpot in assessing anti-AAV immune responses and provides insight into how novel recombinant AAV vectors could be designed to reduce immunogenic potential.
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Krivoshik et al. (2024) studied AAV immune safety. AAV peptide antigens (AAV8 and AAV9 capsid protein sequences) vs. MHC class I or II-specific peptides (positive controls) was evaluated on IFNγ and IL-2-producing cells after AAV peptide presentation. AAV peptide presentation elicited a measurable increase in IFNγ and IL-2-producing cells, with responses varying by AAV serotype and donor, identifying an immunogenic region of the AAV9 capsid.
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