BACKGROUND: Bordetella 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 identify secreted T cell associated cytokines in different age groups after acellular pertussis booster vaccination. MATERIAL AND METHODS: Longitudinal 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 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 (24 h, 48 h, 72 h) were compared. Responses to 15 cytokines were tested with Luminex/multiplex immunoassay. RESULTS: The optimized assay consisted of WB incubation with M, PT, and FHA (including the two co-stimulants). After 48 h incubation, supernatants were collected for measurement of seven selected T cell associated cytokines (IL-2, IL-5, IL-10, IL-13, IL-17 A, 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-17 A/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. CONCLUSIONS: The 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.

Centre for Infectious Disease Control National Institute for Public Health and The Environment Bilthoven The Netherlands

Centre Léon Bérard Lyon France

Clinical Research London School of Hygiene and Tropical Medicine London UK; Institute of International Health Charité Universitätsmedizin Berlin Berlin Germany

Institute of Biomedicine University of Turku Turku Finland

Institute of Biomedicine University of Turku Turku Finland; Department of Paediatrics Turku University Hospital Turku Finland

Laboratory of Medical Immunology Radboud Institute for Molecular Life Sciences Radboud University Medical Center Nijmegen The Netherlands; Radboud Center for Infectious Diseases Radboud University Medical Center Nijmegen The Netherlands

Oxford Vaccine Group Department of Paediatrics University of Oxford Oxford UK

Oxford Vaccine Group Department of Paediatrics University of Oxford Oxford UK; Egas Moniz Center for Interdisciplinary Research Egas Moniz School of Health and Science Almada Portugal

Oxford Vaccine Group Department of Paediatrics University of Oxford Oxford UK; Oxford NIHR Biomedical Research Centre Oxford UK

Sanofi Pasteur Marcy l'Étoile France

Université Libre de Bruxelles Brussels Belgium

Vaccine and Immunity Theme MRC Unit the Gambia Banjul Fajara Gambia

Vaccine and Immunity Theme MRC Unit the Gambia Banjul Fajara Gambia; Clinical Research London School of Hygiene and Tropical Medicine London UK

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Highlights of the 14th International Bordetella Symposium