Edited by: Ramon Arens, Leiden University Medical Center, Netherlands
Reviewed by: Sara Hamilton, University of Minnesota, United States; Rita Carsetti, Bambino Gesù Ospedale Pediatrico (IRCCS), Italy
†Present address: Lotte H. Hendrikx, VU University Medical Center (VUmc), Amsterdam, Netherlands
Specialty section: This article was submitted to Immunological Memory, a section of the journal Frontiers in Immunology
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Pertussis is re-emerging worldwide, despite effective immunization programs for infants and children. Epidemiological studies show a more limited duration of protection against clinical pertussis in adolescents primed with acellular pertussis (aP) vaccines during infancy than those who have been primed with whole-cell pertussis (wP) vaccines. This study aimed to determine whether memory immune responses to aP, diphtheria, and tetanus vaccine antigens following booster vaccinations at 4 and 9 years of age differ between wP- versus aP-primed children.
In a cross-sectional study, blood was collected of DTwP- or diphtheria, tetanus, and aP (DTaP)-primed children before, 1 month, and 2 years after the preschool DTaP booster administered at 4 years of age (
After the preschool booster vaccination, IgG levels were significantly higher in aP-primed as compared with wP-primed children until 6 years of age. Before the preadolescent Tdap booster vaccination, humoral and cellular immune responses were similar in aP- and wP-primed children. However, the Tdap booster vaccination induced lower vaccine antigen-specific humoral, B-cell, and T-helper 1 (Th1) cell responses resulting in significantly lower Th1/Th2 ratios in aP-primed compared with wP-primed children.
The memory immune profiles at preadolescent age to all DTaP vaccine antigens are already determined by the wP or aP combination vaccines given in infancy, showing a beneficial Th1-dominated response after wP-priming. These immunological data corroborate epidemiological data showing that DTaP-primed adolescents are less protected against clinical pertussis than DTwP-primed children.
Since the introduction of whole-cell pertussis (wP) vaccines in the 1940s, widespread vaccination of children strongly reduced the incidence of clinical pertussis (
In a response to the high number of adverse reactions after wP vaccination, acellular pertussis (aP) vaccines have been developed and implemented in the USA from the mid 1990s and subsequently in many national immunization programs (NIP) of high-income countries in the following decades (
The immune mechanisms important for protection against pertussis in humans still remain elusive. Protection is suggested to be mediated by both humoral and cellular immunity (
To elucidate the immune mechanism in relation to long-term protection against pertussis, it is important to evaluate pertussis-specific immune responses over time. In this study, long-term humoral and cellular immune responses to pertussis have been determined in groups of children till adolescent age who received either DTwP or DTaP combination vaccines in infancy following two successive pertussis booster vaccinations at the age of 4 and 9 years.
For this study, blood samples were collected at six different time points from children primed with either wP or aP combination vaccines in the first year of life (Figure
Overview of the study groups. Schematic overview of the six blood sampling time points. Children were primed with either whole-cell pertussis (wP) or acellular pertussis (aP) combination vaccines in the first year of life (2, 3, 4, and 11 months of age) and received a diphtheria, tetanus, and aP (DTaP) booster vaccination at 4 years of age, all according to the Dutch national immunization program. Groups of children were sampled cross-sectionally pre-booster, 1 month and 2 years (at 6 years of age) post DTaP booster vaccination at 4 years of age (indicated in red) (
During infancy, children received either a diphtheria, tetanus, wP, inactivated polio virus,
Blood was sampled at 4 years of age, before the DTaP booster vaccination, 1 month (28 ± 2 days), and 2 years (±2 weeks) after the DTaP booster. From children 9 years of age, blood was sampled just before, 1 month (28 ± 2 days), and 1 year (±2 weeks) after a Tdap booster vaccination. Vacutainer cell preparation tubes containing sodium citrate (Becton Dickinson Biosciences, San Diego, CA, USA) were used for all blood samplings. Peripheral blood mononuclear cells were isolated within 18 h and stored at −135°C, and plasma was stored at −20°C as described (
Plasma IgG antibody concentrations against PT, FHA, Prn, Dd, and tetanus toxin were quantified using the fluorescent bead-based multiplex immunoassay as described (
From a randomly selected subset of 20 longitudinal blood samples of the children aged 9 years, PT, FHA, Prn, and Td-specific enzyme-linked immunospot assays were performed on purified B-cells to determine the numbers of antigen-specific IgG producing memory B-cells (
Geometric mean concentrations with corresponding 95% confidence intervals were calculated for antigen-specific IgG and cytokine responses. Numbers of antigen-specific memory B-cells were counted per 105 B-cells and corresponding geometric mean values were calculated. Normal distribution of (log-transformed) data was checked before analysis. Differences in IgG concentrations between independent or paired samples were tested with corresponding
The study characteristics of wP- and aP-primed children 4–6 years of age and wP-primed children 9 years of age have been described previously (
After the preschool DTaP booster, pertussis-specific IgG levels were significantly higher in aP-primed compared with wP-primed children until 6 years of age (Figure
IgG antibody levels in children 4–9 years of age covering two booster vaccinations.
IgG antibody levels specific for all five vaccine antigens (PT, FHA, Prn, diphtheria, and tetanus) were significantly higher 1 month and 1 year after the preadolescent Tdap booster at 9 years of age compared with pre-booster levels for all children (Table E1 in Supplementary Material). However, we observed differences in dynamics between wP- and aP-primed children. One month after the preadolescent Tdap booster, aP-primed children had significantly lower PT-, FHA-, diphtheria-, and tetanus-IgG levels compared with wP-primed children (all
Reverse cumulative distribution curves of IgG antibody levels in children 9 years of age before and after a Tdap booster vaccination. Reverse cumulative distribution curves of
To exclude possible differences in exposure to pertussis between wP- and aP-primed groups, we compared the IgG levels against PT, which is the specific antigen for
No differences were observed in the number of antigen-specific memory B-cells between wP- and aP-primed children before the preadolescent booster (Figure
Numbers of memory B-cells in children 9 years of age before and after a Tdap booster vaccination. The numbers of
Overall, the number of antigen-specific memory B-cells was significantly higher for all four antigens (PT, FHA, Prn, and tetanus) in both groups at 1 month and 1 year post-booster compared with pre-booster (Figure
We found significantly lower IFN-γ levels in aP-primed compared with wP-primed children for FHA before the booster, for all four antigens (PT, FHA, Prn, and tetanus) at 1 month and for PT, FHA, and tetanus 1 year post-booster (Figure E2 in Supplementary Material), while IL-13 levels only differed for Prn before the booster vaccination between wP- and aP-primed children. Although IL-17 cytokine levels were significantly higher in wP-primed children for FHA and tetanus 1 month post-booster compared with aP-primed children, all IL-17 levels remained low, especially in relation to IFN-γ and IL-13 levels. The IL-10 cytokine levels of vaccine antigen-stimulated T-cells were low and still showed a high variability at all time points (Figure E2 in Supplementary Material).
With higher IFN-γ levels, the ratio of IFN-γ/IL-13 (Th1/Th2 cells) was >1 for all antigens and at all time points in wP-primed children (Figure
T-helper 1 (Th1)/Th2 ratio in children 9 years of age before and after a Tdap booster vaccination. The interferon-gamma (IFN-γ) (Th1)/interleukin-13 (IL-13) (Th2) ratio in supernatants of
No differences in IFN-γ, IL-13, IL-17, and IL-10 cytokine levels were observed between the time points pre- and post-booster within the wP- and aP-primed groups of children, except for lower PT-specific IL-10 production and FHA-specific IL-17 production in wP-primed children 1 year after the booster compared with 1 month (Figure E2 in Supplementary Material).
Based on epidemiological data, waning protection of pertussis-specific immunity seems to occur more rapidly in children who received aP vaccines at infant age in comparison with those who received wP vaccines. In this study, for the first time, we corroborate these epidemiological data with long-term immunological data in groups of children up to 10 years of age showing that priming during infancy with either wP or aP vaccines determines the humoral and cellular immune responsiveness to additional aP booster vaccinations until at least preadolescent age. We clearly showed that replacement of a DTwP combination vaccine by DTaP combination vaccines has enhanced pertussis immune responses on the short term till 6 years of age, 2 years after the fifth DTaP booster at preschool age. However, after the preadolescent Tdap booster vaccination, humoral and cellular immunity showed a shift. While Th2 responses were similar between the two groups, preadolescents primed with wP vaccines during infancy had the more favorable Th1-dominated immune response compared to aP-primed preadolescents for at least 1 year after the booster vaccination. This resulted in a Th2-skewed profile still present in adolescents primed with repeated aP vaccines during infancy that corroborates the increased pertussis incidence in aP-primed adolescents during pertussis outbreaks.
Although the resurgence of pertussis had already started before the introduction of aP vaccines, the incidence of pertussis has sharply increased since aP vaccines have been implemented in all primary and booster vaccination schedules in many countries worldwide (
We showed that the aP-primed children 6 years of age had the advantage of having higher pertussis-specific antibody levels 2 years after the preschool booster compared with wP-primed children. This effect was lost at 9 years of age, with low antibody levels in both wP- and aP-primed children, although Prn-IgG levels remained higher in aP-primed children due to the much higher antibody response shortly after the preschool booster vaccination. Subsequently, the preadolescent booster vaccination induced lower pertussis-specific antibody levels and numbers of memory B-cells in aP-primed compared with wP-primed children. This implies that germinal center responses after wP-priming lead to a more sustainable memory B-cell compartment that is longer boostable during life. Interestingly, we showed that the rise in antibody levels upon a sixth aP vaccination was less than after the fifth aP vaccination, indicating that the degree of antibody production does not persist at the same level despite several vaccine doses. This might be explained by the lower pertussis antigen concentration in the preadolescent Tdap booster vaccination compared with the preschool DTaP booster vaccination. However, wP-primed children, vaccinated with the same DTaP and Tdap booster vaccines as the aP-primed children, showed higher pertussis-specific IgG levels 1 month after the preadolescent booster vaccination compared with 1 month after the preschool booster vaccination. This is in line with Eberhardt and Siegrist, who suggest that germinal center reactions induced by aP-priming vaccines are not effective enough to confer long-term memory responses (
Importantly, the T-cell responses of aP vaccinated children showed a clear Th2-skewed profile, especially after the sixth aP vaccination at 9 years of age for the three pertussis vaccine antigens, as well as for the co-administered tetanus toxoid. Previous studies indicated more Th2 polarization in infants, children, and even in adults who have been primed with aP vaccines compared with wP-priming (
Although Th17 responses have been reported to play an important role in protection against pertussis in mice and baboons (
The recruitment of the two groups of 9-year-old preadolescents was conducted in two different years. This could introduce a different epidemiological background between the cohorts, given the frequent pertussis epidemics in the Netherlands. However, the proportion of PT-IgG seropositive participants [≥50 IU/mL as indication of recent infection (
In conclusion, our pertussis immune responses in preadolescents corroborate the epidemiological data showing that adolescents primed with aP vaccines are less protected against pertussis than those being primed with wP vaccines. New pertussis vaccines should be developed that induce a more Th1-dominated immune response in the primary vaccination series.
This study was carried out in accordance with the recommendations of the medical research ethics committees united (MEC-U, Nieuwegein, the Netherlands) and registered at the European clinical trials database (2013-001864-50) and the Dutch trial register (
SL, LH, GB, and A-MB were involved in the conception, planning, study design, and participant enrollment. SL and LH performed laboratory analysis. SL performed statistical analysis. SL, ES, GB, and A-MB interpreted data and wrote the manuscript. All authors agreed to submit for publication.
SL, LH, GB, and A-MB have no conflicts of interest. ES declares to have received grant support for vaccine studies from Pfizer and GSK.
The authors would like to thank the children and their families for participating in the study, and the nurses of Saltro for performing the clinical procedures. Furthermore, we would like to thank Dr. Tom Wolfs (UMC Utrecht) for acting as the independent physician during this study. From the national institute of public health and the environment (RIVM), we thank Lia de Rond, Kemal Öztürk, Rose-Minke Schure, Simone Leusink, Rianne Boonacker, and Pieter van Gageldonk for their excellent assistance in the measurements of the blood samples, Cécile van Els for her review of the manuscript, and Petra Oomen for recruitment support.
The Supplementary Material for this article can be found online at
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