Citation

BackgroundBordetella pertussis continues to cause whooping cough globally even in countries with high immunisation coverage. Booster vaccinations with acellular pertussis vaccines are thus used in children, adolescents, and adults. T cell immunity is crucial for orchestrating the immune response after vaccination. However, T cell assays can be expensive and difficult to implement in large clinical trials. In this study, a whole blood (WB) stimulation assay was developed to interrogate secreted T cell cytokines in different age groups after acellular pertussis booster vaccination.Material and methodsLongitudinal WB samples were collected from a small set of subjects (n=38) aged 7-70 years participating in a larger ongoing clinical trial. For assay development, samples were real-time diluted and incubated with purified inactivated pertussis toxin (PT), filamentous haemagglutinin (FHA), inactivated B. pertussis lysate, and complete medium (M) as stimulating conditions with anti-CD28 and anti-CD49d as co-stimulants. Different timepoints around the vaccination (D0, D7, D14, D28), WB dilution factor (1:2, 1:4) and incubation time (24h, 48h, and 72h) were compared. Responses to 15 cytokines were tested with Luminex/multiplex immunoassay.ResultsThe optimized assay consisted of WB stimulation with M, PT, and FHA (including the two co-stimulants). After 48h incubation, supernatants were collected for measurement of seven selected T cell signature cytokines (IL-2, IL-5, IL-10, IL-13, IL-17A, IL-17F, and IFN-y) from samples before and 28 days after vaccination. PT stimulation showed a trend for upregulation of IL-2, IL-13, and IL-17A/F for adult subjects, whereas the responses of all cytokines were downregulated for the paediatric subjects. Furthermore, PT and FHA-stimulated WB showed diverse cytokine producing profiles.ConclusionsThe developed WB-based cytokine assay was shown to be less costly, easy to perform, and functional in differently aged individuals. Further, it requires only a small amount of fresh blood, which is beneficial especially for studies including infants. Our results support the use of this assay for other immunological studies in the future.