(M0930-02-10) Multiplexed Cytokine Measurements in Culture Supernatants upon Ex Vivo Stimulation of Human PBMCs to Assess Cellular Immune Response in Vaccine Studies

Purpose: Vaccine induced immune responses can be broadly characterized into humoral (antibody) and cell mediated immune (CMI) responses. Functional or neutralizing antibodies have long been a key measure to assess how immunogenic a vaccine is and if it is likely to provide protection from natural infection upon exposure. Recent findings suggest that the T-cell-mediated immune response also plays a crucial role in vaccine effectiveness. T cells can have direct action against pathogens, are potent producers of cytokines and factors that drive protective immune responses and support B-cell maturation and antibody production. Moreover, vaccines that can stimulate the production of memory B cells and T cells are key to providing durable immunity. However, fully comprehending the specific contributions of CMI responses to vaccine-induced immunity is challenging, partly due to the difficulty in obtaining sufficient immune cell samples for analysis in clinical trials. To address this, we introduce the Activation-Induced Marker (AIM) multiplex cytokine assay, a sensitive method designed to profile the CMI response in vaccine research and clinical studies. This assay, which evaluates the antigen recall response, leverages the ex-vivo stimulation of Peripheral Blood Mononuclear Cells (PBMCs) and the subsequent measurement of cytokines in the supernatant to serve as a functional read-out. The quantity and variety of cytokines detected using PBMCs during vaccine clinical trials will provide insight into the magnitude and nature of the immune response following immunization.

Methods: Cryopreserved human PBMCs were thawed and rested overnight in culture at 37°C. Rested cells were washed, counted, and adjusted to plate 0.5 ×10⁶ viable cells per well. Cells were then stimulated ex-vivo with a pool of peptide antigens representing the antigens in the vaccine for twenty-four hours. In addition, PBMCs were incubated with an equimolar amount of DMSO as negative control, CEFX peptide pool as a low positive control, and with Staphylococcal Enterotoxin B (SEB, 0.5 mg/ml) as a positive control. After stimulation, supernatants were harvested for cytokine detection by Protein Simple’s automated immunoassay platform ELLA using a qualified custom-built 8-plex cytokine panel. Qualification of the ELLA 8-plex cytokine panel that includes both Type 1 and Type 2 cytokines (i.e. IL-2, IL-4, IL-5, IL-10, IL-13, IFN-g, TNF-a, and CXCL-10) was performed following United States Pharmacopeia Chapter 1033 Biological Assay Qualification and ICH M10 Bioanalytical Method Validation and Study Sample Analysis guidelines. Briefly, assay qualification parameters included assessment of Precision, Parallelism, Linearity, Sensitivity, Specificity, and Stability.

Results: Method qualification results for the multiplex assay met all pre-specified acceptance criteria. For the precision assessment, a panel of 26 PBMC culture supernatant samples spanning the range of the assay were tested at the minimum required dilution (MRD) of 1:2 utilizing different lots of assay cartridges by two analysts on three different days generating 6 assay runs in total. The assay demonstrated acceptable precision with repeatability and intermediate precision spanning from 6.3% to 17.4% (precision acceptance criteria ≤25%CV). For the parallelism assessment, a total of 12 samples including CEFX and SEB stimulated, and recombinant cytokine spiked PBMC culture supernatants were serially diluted (2-fold) starting at MRD to 1:64 fold. The assay demonstrated acceptable parallelism with relative accuracy ≤25%. Linearity was assessed by testing a panel of 6 samples at 8 to 11 dilutions to reach the bottom of the assay ranges. Sensitivity (Lower Limit of Quantification, LLOQ) was defined as the lowest concentration where the assay accurately and precisely measures an analyte. Both LLOQ and Upper Limit of Quantification (ULOQ) encompassing the analytical measurement range met linearity and precision acceptance criteria. Single digit pg/mL level sensitivity (LLOQ) was achieved for all 8 cytokine assays, whereas ULOQ ranged from 3 to 5-digit pg/mL levels. Specificity was assessed by spiking all 8 recombinant cytokines in a PBMC culture supernatant sample and determining % recovery (%specificity) for individual cytokines. The percentage recovery of each cytokine was within 75 to 125% meeting the specificity requirement and confirming no assay cross-reactivity. Sample stability was assessed up to three freeze-thaw cycles and 40 days storage at -60°C to -90°C using 4 to 6 stimulated PBMC supernatant samples. The samples demonstrated acceptable stability presenting %recovery compared to baseline within the 75% to 125% range.

Conclusion: We have developed a robust, precise, and sensitive assay with a wide dynamic range for measuring the CMI response specific to antigens, focusing on cytokine production as the read-out of the activation state of PBMCs. The assay, which has been validated for use with 0.5 × 10⁶ viable cells per stimulation condition, enables the detailed characterization of Type 1 and Type 2 cytokine production profiles in reaction to vaccine antigens. This method will be employed to evaluate the CMI responses elicited by Moderna's vaccine candidates in the context of clinical research.