Background: Pertussis can lead to serious disease and even death in infants. Older adults are more vulnerable to complications as well. In high-income countries, acellular pertussis vaccines are used for priming vaccination. In the administration of booster vaccinations to different age groups and target populations there is a substantial between-country variation. We investigated the effect of age on the response to acellular pertussis booster vaccination in three European countries. Methods: This phase IV longitudinal intervention study performed in Finland, the Netherlands and the United Kingdom between October 2017 and January 2019 compared the vaccine responses between healthy participants of four age groups: children (7À10y), adolescents (11À15y), young adults (20À34y), and older adults (60À70y). All participants received a three-component acellular pertussis vaccine. Serum IgG and IgA antibody concentrations to pertussis antigens at day 0, 28, and 1 year were measured with a multiplex immunoassay, using pertussis toxin concentrations at day 28 as primary outcome. This trial is registered with ClinicalTrialsRegister.eu (2016À003,678À42). Findings: Children (n = 109), adolescents (n = 121), young adults (n = 74), and older adults (n = 75) showed high IgG antibody concentrations to pertussis toxin at day 28 with GMCs of 147 (95% CI 120À181), 161 (95% CI 132À196), 103 (95% CI 80À133), and 121 IU/ml (95% CI 94À155), respectively. A significant increase in GMCs for vaccine antigens in all age groups by 28 days was found which had decreased by 1 year. Differences in patterns of IgG GMCs at 28 days and 1 year post-vaccination did not have a consistent relationship to age. In contrast, IgA antibodies for all antigens increased with age at all timepoints. Interpretation: Acellular pertussis booster vaccination induces significant serum IgG responses to pertussis antigens across the age range which are not uniformly less in older adults. Acellular boosters could be considered for older adults to reduce the health and economic burden of pertussis.

To advance research and development of improved pertussis vaccines, new immunoassays are needed to qualify the outcome of Bordetella pertussis (Bp) specific CD4+ T-cell differentiation. Here, we applied a recently developed whole blood assay to evaluate Bp specific CD4+ T-cell responses. The assay is based on intracellular cytokine detection after overnight in vitro Bp antigen stimulation of diluted whole blood. We show for the first time that CD4+ T-cell memory of Th1, Th2, and Th17 lineages can be identified simultaneously in whole blood. Participants ranging from 7 to 70 years of age with different priming backgrounds of whole-cell pertussis (wP) and acellular pertussis (aP) vaccination were analyzed around an acellular booster vaccination. The assay allowed detection of low frequent antigen-specific CD4+ T-cells and revealed significantly elevated numbers of activated and cytokine-producing CD4+ T-cells, with a significant tendency to segregate recall responses based on primary vaccination background. A stronger Th2 response hallmarked an aP primed cohort compared to a wP primed cohort. In conclusion, analysis of Bp specific CD4+ T-cell responses in whole blood showed separation based on vaccination background and provides a promising tool to assess the quantity and quality of CD4+ T-cell responses induced by vaccine candidates.

Pertussis is a vaccine-preventable disease caused by the bacterium Bordetella pertussis. Over the past years, the incidence and mortality of pertussis increased significantly. A possible cause is the switch from whole-cell to acellular pertussis vaccines, although other factors may also contribute. Here, we applied high-dimensional flow cytometry to investigate changes in B cells in individuals of different ages and distinct priming backgrounds upon administration of an acellular pertussis booster vaccine. Participants were divided over four age cohorts. We compared longitudinal kinetics within each cohort and between the different cohorts. Changes in the B-cell compartment were correlated to numbers of vaccine-specific B- and plasma cells and serum Ig levels. Expansion and maturation of plasma cells 7 days postvaccination was the most prominent cellular change in all age groups and was most pronounced for more mature IgG1+ plasma cells. Plasma cell responses were stronger in individuals primed with whole-cell vaccine than in individuals primed with acellular vaccine. Moreover, IgG1+ and IgA1+ plasma cell expansion correlated with FHA-, Prn-, or PT- specific serum IgG or IgA levels. Our study indicates plasma cells as a potential early cellular marker of an immune response and contributes to understanding differences in immune responses between age groups and primary vaccination backgrounds.

Innate myeloid cell (IMC) populations form an essential part of innate immunity. Flow cytometric (FCM) monitoring of IMCs in peripheral blood (PB) has great clinical potential for disease monitoring due to their role in maintenance of tissue homeostasis and ability to sense micro-environmental changes, such as inflammatory processes and tissue damage. However, the lack of standardized and validated approaches has hampered broad clinical implementation. For accurate identification and separation of IMC populations, 62 antibodies against 44 different proteins were evaluated. In multiple rounds of EuroFlow-based design-testing-evaluation-redesign, finally 16 antibodies were selected for their non-redundancy and separation power. Accordingly, two antibody combinations were designed for fast, sensitive, and reproducible FCM monitoring of IMC populations in PB in clinical settings (11-color; 13 antibodies) and translational research (14-color; 16 antibodies). Performance of pre-analytical and analytical variables among different instruments, together with optimized post-analytical data analysis and reference values were assessed. Overall, 265 blood samples were used for design and validation of the antibody combinations and in vitro functional assays, as well as for assessing the impact of sample preparation procedures and conditions. The two (11- and 14-color) antibody combinations allowed for robust and sensitive detection of 19 and 23 IMC populations, respectively. Highly reproducible identification and enumeration of IMC populations was achieved, independently of anticoagulant, type of FCM instrument and center, particularly when database/software-guided automated (vs. manual “expert-based”) gating was used. Whereas no significant changes were observed in identification of IMC populations for up to 24h delayed sample processing, a significant impact was observed in their absolute counts after >12h delay. Therefore, accurate identification and quantitation of IMC populations requires sample processing on the same day. Significantly different counts were observed in PB for multiple IMC populations according to age and sex. Consequently, PB samples from 116 healthy donors (8-69 years) were used for collecting age and sex related reference values for all IMC populations. In summary, the two antibody combinations and FCM approach allow for rapid, standardized, automated and reproducible identification of 19 and 23 IMC populations in PB, suited for monitoring of innate immune responses in clinical and translational research settings.

BackgroundImmunogenicity of acellular pertussis (aP) vaccines is conventionally assessed by measuring antibody responses but antibody concentrations wane quickly after vaccination. Memory B cells, however, are critical in sustaining long-term protection and therefore may be an important factor when assessing pertussis immunity after vaccination.AimWe studied pertussis specific memory B cell (re)activation induced by an aP booster vaccination in four different age groups within three countries.Materials and methodsFrom a phase IV longitudinal interventional study, 268 participants across Finland, the Netherlands and the United Kingdom were included and received a 3-component pertussis booster vaccine: children (7-10y, n=53), adolescents (11-15y, n=66), young adults (20-34y, n=74), and older adults (60-70y, n=75). Memory B cells at baseline, day 28, and 1 year post-vaccination were measured by a pertussis toxin (Ptx), filamentous haemagglutinin (FHA), and pertactin (Prn) specific ELISpot assay. Antibody results measured previously were available for comparison. Furthermore, study participants were distributed into groups based on their baseline memory B cell frequencies, vaccine responses were monitored between these groups.ResultsGeometric mean (GM) memory B cell frequencies for pertussis antigens at baseline were low. At 28 days post-vaccination, these frequencies increased within each age group and were still elevated one year post-booster compared to baseline. Highest frequencies at day 28 were found within adolescents (GM: 5, 21, and 13, for Ptx, FHA and Prn, respectively) and lowest within older adults (GM: 2, 9, and 3, respectively). Moderate to strong correlations between memory B cell frequencies at day 28 and antibody concentrations at day 28 and 1 year were observed for Prn. Memory B cell frequencies > 1 per 100,000 PBMCs at baseline were associated with significantly higher memory responses after 28 days and 1 year.ConclusionsAn aP booster vaccine (re)activated memory B cells in all age groups. Still elevated memory B cell frequencies after one year indicates enhanced immunological memory. However, antigen specific memory B cell activation seems weaker in older adults, which might reflect immunosenescence. Furthermore, the presence of circulating memory B cells at baseline positively affects memory B cell responses. This study was registered at www.clinicaltrialsregister.eu: No. 2016-003678-42.