(T1530-02-08) Development and Fit-for-Purpose(FFP) Validation of a 35-Color Ex Vivo Stimulation and Activation-Induced Marker (AIM) Assay to Assess Vaccine-Induced Immune Responses in Human Vaccine Trials

Purpose: Both humoral and cell-mediated immune responses are induced by vaccines. Cell-mediated immune responses not only have direct cell action against pathogens, but also support the humoral immune response. Activation of the T cell compartment plays a critical role in long lasting protection by inducing immune memory. Activation-induced marker (AIM) assays are a suitable option to assess cell- mediated immune responses (CMI). Antigen-specific T cell detection requires ex vivo stimulation of peripheral blood mononuclear cells (PBMCs) with antigens of interest where autologous antigen-presenting cells process and present peptide in complex with major histocompatibility complex (MHC) molecules. T cell receptors (TCR) recognize these peptide-MHC complexes, resulting in upregulation of activation markers on T cells that can be detected by flow cytometry (Poloni et al. 2023). To address exploratory endpoints in the clinical study, we developed and validated a fit-for-purpose 35-color AIM assay to monitor antigen specific T-cell populations along with immunophenotyping of other lymphoid and myeloid populations.

Methods: Cryopreserved PBMCs are thawed and rested overnight in culture at 37°C. Rested cells are washed, counted, and adjusted to plate 0.5 x 10⁶ viable cells per well. Cells are then stimulated with vaccine specific peptide pools for 24 hours. After stimulation, cells are collected, washed, and stained with surface marker antibodies and with viability dye. Cells are washed and fixed with 4% PFA for 30 mins. Finally, each sample is filtered to remove clumped cells and acquired on a Cytek Aurora. Data files are analyzed using FCS Express IVD software (DeNovo Software, V7.0). Validation method was conducted following CLSI H-62 guidelines. It includes Assay Precision assessments of Repeatability (Intra-Assay) and Reproducibility (Inter-Instrument, Inter-Analyst, Inter-Run), and post-staining sample stability.

Results: An acceptance criterion of ≤ 25% CV was applied for intra-assay, inter-assay, inter-analyst, and inter-instrument precision. Stability was established at the latest time point where a ≤ 25% change from the baseline was observed. AIM+ CD4 T cells subsets (CD69+OX40+,  CD69+CD40L+, CD25+OX40+, CD25+PD-L1+, OX40+CD137+, and CD69+CD137+), AIM+ CD8 T cell subsets (CD69+CD137+ and CD25+PD-L1+), and AIM+ circulating T follicular helper cells, cTfh (CD4+CXCR5+OX40+ CD40L+) passed intra-assay, inter-assay, inter-analyst, and inter-instrument precision. Moreover, all these AIM populations also passed fixed sample stability test up to 24 hours.

Conclusion: We have established a robust and performing assay that allows for the detection of antigen-specific T cells by assessing CD69, OX40, CD137, CD25, PD-L1, and CD40L. This assay will be used to assess cell mediated immune responses induced by Moderna trail vaccines.

References: Poloni C, Schonhofer C, Ivison S, Levings MK, Steiner TS, Cook L. T-cell activation-induced marker assays in health and disease. Immunol Cell Biol. 2023 Jul;101(6):491-503.

Acknowledgements: All authors are employees of Moderna, Inc. and may own stock/stock options in the company. This study was funded by Moderna, Inc.