The present study represents the first reported evaluation of T-cell responses upon a second acellular pertussis booster vaccination at the pre-adolescent age, 9 years of age, and 5 years after a preschool booster vaccination in children 4 years of age. The second aP booster vaccination at pre-adolescent age in wP primed individuals did increase pertussis-specific Th1 and Th2 cytokine responses. However, almost all T-cell responses were already high before the booster vaccination at 9 years of age and had enhanced significantly during the 5-year period between the two booster vaccinations. Higher T-cell booster responses were also observed 1 month after the extra booster vaccination at 9 years of age than 1 month after the preschool booster vaccination at 4 years of age. The fact that T-cell responses at 9 years of age were higher than those directly after the preschool booster at 4 years and at 6 years of age implies that natural boosting has stimulated these T-cell responses, which can be explained by the high circulation of pertussis in the Netherlands 
. This seems in line with the presence of higher numbers of PT-specific (proliferated) effector memory CD4+ T-cells in children 9 years compared to those at 4 years of age.
In the Netherlands, an increased, high circulation of pertussis was found in the last decade as shown by carefully monitoring the incidence of pertussis as well as the evaluation of pertussis IgG levels in persons from 0 till 80 years of age in two large cross-sectional serosurveillance studies performed in 1995/6 and 2006/7 
. Also, the incidence of pertussis has shifted from young to older children since the implementation of the preschool aP booster vaccination at 4 years of age 
. Very recent data have shown that the positive effect of the preschool booster lasts until the age of 13. Therefore, nowadays the increased incidence of whooping cough is mainly found in adolescents and adults 
It is difficult to compare our study with other studies on cell-mediated immune responses against pertussis and the effects of booster vaccinations, because each study used different age groups of children, vaccination schedules or measured different T-cell parameters 
. In contrast to other studies 
, we have shown just a small increase of pertussis-specific T-cell immunity induced by a second booster at pre-adolescent age, because T-cell immunity was already elevated in these 9 years old children. The limited increase and the discrepancy with other publications describing a longer persistence of cell-mediated immune responses after booster vaccination 
can be explained by the effect of a preschool booster vaccination 5 years earlier, at 4 years of age, in combination with boosting by natural infection in our study cohort.
Our results additionally showed higher numbers of pertussis-specific CD3+CD4+ effector memory T-cells in 9 years old children compared with 4 years old children. Although the percentages of effector memory T-cells producing Th1 cytokines between 4 and 9 years old children were similar, the higher number of these cells in older children resulted into higher amounts of Th1 cytokines. Notably, in both groups of children, CD3+CD4+ effector memory cells producing simultaneously IFN-γ and TNF-α upon stimulation with pertussis antigens were found, indicating that one cell is able to produce more than 1 cytokine 
The concentrations of T-cell cytokines in 9 years old children both before and at 1 month after booster vaccination predominantly showed Th1 IFN-γ responses and fewer IL-13 responses, associated with Th2 cytokine lineage, or IL-17 responses. Therefore, particularly in children, Th1 responses might play an important role in protection against clinical pertussis infection, while in general IL-17 was suggested to play a more prominent role in older individuals 
Phenotypical characterization of T-cells in adolescents by Rieber et al. after an aP booster following wP or aP priming at infancy confirmed a predominant Th1 response especially by activation of CD8 T-cells 
. However, in contrast to these results, we found a higher percentage of CD4 effector memory T-cells (CCR7+CD45RA-) than CD8 effector memory cells in the children in our study. Next to pertussis-specific CD8+ memory T cells that may contribute to protection against clinical pertussis 
, we believe that also CD4+ T cells producing Th1-cytokines may play an important role in protection of children against clinical pertussis.
We recently showed increased pertussis-specific memory B-cell immune responses after the second aP booster vaccination in Dutch wP primed children 9 years of age that sustained at least for one year 
. The correlation between the high numbers of pertussis-specific memory B-cells at one month after this second booster vaccination with the corresponding antibody responses still present after one year indicates the important role of the memory B-cell pool in the maintenance of antibody levels. A second aP booster vaccine affects B-cell memory more than the T-cell memory responses. We speculate that a certain level of T-helper-cell memory immunity is needed to be able to increase the B-cell responses upon booster vaccination. Epidemiological data reveal an improved protection against pertussis after the implementation of aP booster vaccines at 4 years of age 
. These studies together with the increased T-cell and B-cell responses upon the second aP-booster vaccinations at pre-adolescent age, which is strengthened by the circulation of pertussis, might result into a better protection against pertussis during adolescence. For a better understanding of the duration of pertussis-specific memory immunity, this needs to be monitored over the longer term.
Although other West European countries had already switched from wP to aP infant vaccinations in the 1990s, pertussis still circulates among these aP primed populations too and notable pertussis incidences have been equally observed in these countries as in The Netherlands that implemented the vaccine switch only in 2005. Meanwhile, concerns about the efficacy of repetitive aP booster vaccinations have risen 
. In Canada, low vaccination coverage was observed 5 years after the implementation of adult booster vaccinations 
. Moreover, repeated booster vaccinations in adults in general are not considered cost-effective 
, since the yet unvaccinated newborns are particularly the risk group susceptible for severe infection. As transmission studies have shown, mothers are one of the main sources of infection for the young infant 
. Thus although natural boosting of the population will also provide pertussis-specific immune responses that improve protection, the vulnerable unprotected young infant is put at risk by a high incidence of pertussis in the adult population. This means that selective vaccination of those adults who are in close contact with infants, the so-called cocooning strategy, will better reduce transmission to infants and will probably be more (cost)-effective 
Because a majority of the Dutch population born before 1997 has only received the Dutch wP vaccine during infancy, we believe that aP booster vaccination in this part of the population will improve long-term immunity against pertussis substantially. Such a vaccine strategy might enlarge also the antibody levels in woman at childbearing age, in turn increasing the maternal transfer of antibodies to newborns who subsequently will be better protected. However, pertussis booster vaccinations in adults are expensive and the vaccination coverage in the adult population has proven to be generally low 
. Therefore, a good information and communication strategy must accompany such a booster vaccination in order to make its implementation in the late adolescent or young adult population successfully.
In conclusion, we demonstrated, in Dutch wP primed children 9 years of age, an enhanced Th1 memory immune response upon a second aP booster vaccination. These results, together with enhanced memory B-cell responses upon booster vaccination support the introduction of an aP booster vaccination for preadolescents. The positive effect of the preschool aP booster vaccination at age 4 years in combination with natural boosting of the immune responses by circulation of pertussis will probably protect wP-primed children until teenage. An aP booster vaccination at adolescence or later might improve long-term immunity against pertussis and reduce the transmission to the vulnerable newborns.