Antibody waning and Bordetella pertussis resurgence after the COVID-19 pandemic in the Netherlands

Domain: chemistry
Published: 2026-02-20
Editorial outcome: accept

Abstract
After relative absence of Bordetella pertussis during the COVID-19 pandemic, notifications in the Netherlands were increased from May 2023 till September 2024. We monitored the largest pertussis outbreak in decades and investigated underlying immunological dynamics based on IgG and IgA antibodies against pertussis antigens (Ptx, FHA & Prn). We analyzed serum from 418 participants (2–87 years) at five time points (November 2022-October 2024) from a nationwide prospective serosurveillance study (PIENTER-Corona). Weighted incidence over two years was 6.3% (95% CI: 4.4-8.2) in the Dutch population, and 35% (95% CI: 26.2-44.6) in 6-18 y-olds. Children aged 6-12 years, who received a booster vaccination at 4 years, showed lowest anti-Ptx IgG concentrations after the period of reduced circulation. Infected individuals had lower pre-infection anti-FHA IgG and IgA concentrations than matched uninfected individuals. Prolonged coughing was reported by 16% of the infected individuals. Our findings highlight the B. pertussis outbreak was preceded by waning immunity, particularly after acellular pertussis booster vaccination. The small proportion of symptomatic infections implies sufficient protection by the vaccine against disease but not transmission.

Introduction
During the COVID-19 pandemic, non-pharmaceutical interventions (NPIs), including wearing face masks, social distancing, closure of schools and working from home, were introduced to limit the circulation of SARS-CoV-2. This not only led to a reduction of SARS-CoV-2 notifications but also to a decrease of notifications of other airborne-transmissible pathogens, including pertussis, or whooping cough, a disease caused by the bacterium Bordetella pertussis (Fig. 1)1–3. Notably, this reduction of pertussis notifications likely reflects reduced circulation of the bacterium, which lasted for almost 3 years. Since May 2023, pertussis notifications rose, cumulating in the largest pertussis outbreak to date since 2012 in the Netherlands (Fig. 1)2. This trend has been observed in multiple Europe countries2,3.

Pertussis is a highly contagious respiratory infection. During the catarrhal stage, symptoms mostly resemble those of a common cold. In the paroxysmal phase, which is the most symptomatic stage, symptoms are often coughing, inspiratory whooping and post-tussive vomiting4. Infections can occur at any age but can cause serious disease in (predominantly unvaccinated) infants and older adults4. Vaccinated individuals are more likely to have an atypical, mild, or even asymptomatic infection5. Since the use of the whole cell pertussis (wP) vaccine—containing inactivated bacteria—in the Dutch national immunization program (NIP) in 1957, a drop in the incidence of pertussis was recorded6. Despite vaccination, re-emergence was apparent as of 1996, with incidence peaking every 3–5 years7. In 2001, an acellular pertussis (aP) booster vaccination, containing inactivated pertussis toxin (Ptx), filamentous hemagglutinin (FHA), pertactin (Prn) and sometimes fimbriae 2/3 (Fim 2/3), was implemented at 4 years of age2. In 2005, the wP vaccine for the primary series was also replaced by an aP vaccine due to high reactogenicity of the wP vaccine2. Despite these changes, pertussis incidence remained high and a threat for infants too young to be vaccinated2. Of special concern is the decrease in vaccination coverage in the Netherlands that has been observed over the past years. The WHO and RIVM reported that the percentage of fully vaccinated infants by the age of two declined from 96 to 97% in the 2010 birth cohort to 92–94% in the 2019 birth cohort and 89–93% in the 2022 birth cohort8,9. Since 2019, the maternal diphtheria-tetanus-acellular pertussis (Tdap) vaccination is implemented in the Dutch NIP to increase the level of maternally-derived immunological protection in the first months post birth2. However, this is not implemented globally, and pertussis remains one of the least controlled vaccine-preventable diseases.

As B. pertussis generally circulates continuously, leading to notifications year-round, there is no clear understanding of what drives the epidemiology and the elevation of notifications every 3–5 years. Notably, while respiratory syncytial virus and seasonal coronaviruses re-appeared rapidly when most NPIs for COVID-19 were lifted mid-2022, pertussis notifications increased nearly a year later (Fig. 1). The period of reduced B. pertussis circulation provided a unique opportunity to monitor waning immunity against B. pertussis and how changes in immunity may have affected the rapid increase of notifications from 2023 onwards. We use a Dutch nationwide prospective cohort (2–87 year of age), setup since the beginning of the COVID-19 pandemic, to investigate the age-specific incidence of B. pertussis infections between November 2022 and October 2024. We identify an overall 6.4% incidence, and an incidence of 35.4% in children aged 6–18 years during this two-year period, which is caused by the combined effect of waning vaccine immunity and a prolonged period of reduced pertussis circulation.

Results
Study population
The participants for this study were part of the PICO cohort and resided across the country (Supplementary Fig. 1A). Slightly more women were included compared to men (Table 1). Vaccination coverage in children based on long-term protection anti-Ttx IgG (>0.1 IU/ml) was estimated to be 98.9% at baseline (i.e., November 2022). Overall, anti-Ttx IgG antibodies were detected in 95.2% of the included participants.

Overall anti-Ptx IgG concentrations by age were determined at the start and end of the study (Fig. 2A). At the start, lower concentrations were observed in 6–12-year-old individuals as compared to 2–5-year-olds and adults (p < 0.005). Anti-Ptx IgG concentrations of 6–12-year-old individuals had increased in October 2024 as compared to those at the start of the study (p < 0.001), despite large variation (Fig. 2B). In all other age groups, no significantly different concentrations were observed between both sampling rounds (Fig. 2B).

Pertussis infections during the study period
Antibody rises attributed to DTaP or Tdap vaccination during the study period were observed in the 2–5 years age group (13 cases) and in the 19–39 years age group (4 cases) (Fig. 3A, blue lines). The weighted incidence of B. pertussis infections in the general Dutch population—based on the serological criteria in the current study—was 0.6% between November 2022 and April 2023, 1.4% between April and November 2023, 3.5% between November 2023 and April 2024, and 1.5% between April and October 2024 (Table 1 and Fig. 3A, orange lines). The weighted cumulative incidence in the Dutch population (2–87 years) during this two-year period was estimated at 6.3%. The highest incidence (35.4%) was found among the age group 6–18 years (Fig. 3B and Table 1). The increase in anti-Ptx IgG concentrations after infection was also paralleled by an increase in anti-FHA and anti-Prn IgG and IgA and anti-Ptx IgA concentrations (Supplementary Fig. 3).

Individuals with serological evidence of B. pertussis infection did not report increased symptoms compared to uninfected individuals. Prolonged coughing (>2 weeks) during the past year was reported by 16% of the infected individuals between 6 and 18 years (5/32) in the last sampling round, of whom two contacted the GP. No significant difference was observed concerning prolonged coughing in uninfected individuals (22/75 (29.3%); p = 0.21).

Because the higher pertussis incidence according to serological detections, including age group differences (Table 1), and the syndromic notifications (Supplementary Fig. 1B), the serological infection prevalence based on anti-Ptx IgG categories was further estimated in an additional subset in October 2024. The additional cross-sectional subset showed a similar proportion of individuals with no indication of recent infection (<50 IU/mL) in the 6–18, 19–39, and 60+ year age groups compared to the longitudinal cohort (Supplementary Fig. 4). The 40–59 years age group indicated a lower proportion of recently infected individuals in the longitudinal serosurvey in October 2024 compared to the cross-sectional study (p = 0.0028) (Supplementary Fig. 4).

The cross-sectional subset from October 2024 was also compared with previous cross-sectional serosurveys conducted using similar methodologies7. All age groups show a similar proportion of individuals with no indication of recent infection compared to the Pienter-2 study (2006–2007) and the Pienter-3 study (2016–2017) except for individuals in the 40–59 years age group (Fig. 4). The additional subset from October 2024 showed a higher proportion of individuals without indication of recent infection compared to the Pienter-3 study (p = 0.045).

Antibody waning
To address whether the lower anti-Ptx IgG concentrations within the 6–18 years age group in November 2022 (Fig. 2) might be related to a more rapid antibody waning after (primary series or booster) vaccination, a linear mixed-effects model was applied in individuals who remained uninfected or up the point of infection and unvaccinated. The 2–5 year-olds showed the highest anti-Ptx IgG concentrations at the start of the study (16.45 IU/mL; p < 0.05 compared to other age groups). The 6–12 years age group displayed lower antibody concentrations at the start of the study period compared to adults (5.83 IU/mL vs. 7.28 IU/mL; p = 0.009). The fastest antibody decay was observed in the 2–5 years age group with the duration of antibody decay to 50% of 17.8 months (Fig. 5; p < 0.001 for the slope). Antibody decay was also noticed in the 6–12, 13–18 and ≥19 years age group (p < 0.001 for the slopes). The duration of antibody decay to 50% in the 6–12, 13–18, and ≥19 years age group was 48.5, 102.5 and 84.1 months, respectively.

Anti-Ptx, FHA and Prn concentrations in individuals 6–18 years
Anti-FHA IgG concentrations of infected individuals between 6 and 18 years of age appeared significantly lower prior to infection compared to age-matched uninfected individuals (13.4 IU/mL (95% CI 9.8–18.2) vs. 27.9 IU/mL (95% CI 22.1–35.1); p = 0.002), as well as anti-FHA IgA concentrations (1.0 IU/mL (95% CI 0.8–1.4) vs. 2.1 IU/mL (95% CI 1.6-2.8); p = 0.006) (Fig. 6B, E). Comparison of anti-Ptx, anti-Prn IgG, and IgA concentrations revealed no statistically significant differences, nor did the control antigen rubella IgG concentrations.

Using the Youden index, protective cut-off values for anti-FHA IgG and IgA were estimated. This resulted in cutoffs of 13.6 and 1.6 IU/mL for IgG and IgA respectively, with corresponding AUC values of 0.74 and 0.72 (specificity of 78% and 63% and sensitivity of 71% and 74%, respectively) (Supplementary Fig. 5). We found no conclusive evidence that individuals who reporting (prolonged) cough symptoms had statistically significant lower anti-Ptx IgG and IgA concentrations, although the numbers were low (p = 0.084 and p = 0.051, respectively).

Methods
Study population
The PIENTER-corona (PICO) study was setup at the start of the COVID-19 pandemic to study immunity to SARS-CoV-2 across the Dutch population with repeated sampling of the same individuals (~10,000 unique participants) until the end of 2024 (in total 13 study rounds), as described previously37,40,41. The PICO-study protocol was approved by the Medical Ethics Committee MEC-U, the Netherlands (Clinical Trial Registration NTR8473), and conformed to the principles embodied in the Declaration of Helsinki. Informed consent was obtained from all adult participants and from parents or legal guardians of minors prior to participation.

Here, we randomly selected an age-stratified subset with completed questionnaires and samples (n = 419) from study rounds 9–13 (November 2022–October 2024) (age range: 2–87 years). Because of the vaccination schedule and expected incidence in children, smaller age windows were used (Table 1). For the overall analysis, we expected a 6% incidence rate7 and aimed for a one-sided precision of 2.5%, resulting in a minimum required sample size of 347 individuals (α = 0.05, power = 80%). Taking into account a potential loss of 15% of follow-up samples, at least 408 participants were needed in the longitudinal analysis. For all age groups, we were able to include sufficient individuals, except for the youngest age group (2–5 years), where only 43 individuals were available, who were all included.

To investigate changes in antibody concentrations, samples were collected in November 2022 (median inclusion date: 8 November), April 2023 (median: 25 April), and November 2023 (median: 6 November) (Supplementary Fig. 1B). Sampling volumes were adequate for all but one participant, and serum samples from one participant had to be excluded due to technical issues, so serum samples from 418 participants could be tested. Since the pertussis notifications were still elevated after November 2023, follow-up samples collected in April 2024 (median: 15 April; 382 participants (91%)) and October 2024 (median: 14 October; 358 participants (85%)) were included as well.

Participants sent in a fingerstick blood sample in a microtainer by mail. Serum obtained from these blood samples was stored at −20°C awaiting analyses. The participants completed a questionnaire during each sampling round, including, amongst others, questions about sociodemographic characteristics, COVID-19-related symptoms (general, respiratory, and gastrointestinal) in the past months and behavioral factors. Each round, participants were requested to report respiratory symptoms, including cough, sore throat and runny nose and fever, which are associated with B. pertussis infection24. In the final sampling round, a specific question about prolonged coughing (>2 weeks) in the past year was added.

Detection of antibodies
Serum samples were tested for anti-Ptx, anti-tetanus toxin (Ttx; to control for vaccination) and anti-rubella (as an independent control to monitor antibody variation) IgG antibodies at all sampling rounds using an in-house validated fluorescent bead-based multiplex immunoassay as described before42,43. During the last three sampling rounds, most infections were detected, hence, these samples were tested additionally for anti-FHA and anti-Prn IgG and IgA and Ptx IgA (see Supplementary Methods for further details on the assay).

Criteria for pertussis infection
Active or recent infection with B. pertussis was detected by an increase in Ptx-specific antibodies44. Diagnostic confirmation of recent pertussis infection in paired samples, with the second sample at 2–4 weeks interval, normally uses a >3-fold increase in anti-Ptx IgG concentration with a final concentration >20 IU/mL44–46. Here, we used a ≥2-fold increase criterion due to a longer time interval between two measurements. We expected more waning of antibodies within that period, consequently leading to a lower fold-change between consecutive samples. For participants of whom a sample was missing in November 2022 (n = 1) or April 2024 (n = 11), an infection could be ruled out as they remained below the cutoff in the subsequent round (April 2023 or October 2024).

Part of the participants in this study could have elevated anti-Ptx concentrations due to DTaP or Tdap infant, booster or maternal vaccination (Table 2). For children <5 years of age and women who reported to be pregnant, a ≥2-fold increase in anti-Ptx IgG and anti-Ttx IgG concentrations with anti-Ptx IgG concentrations >5 IU/mL indicated a recent (maternal) vaccination. Because the Tdap booster vaccination contains reduced concentrations of the pertussis components, and vaccination induces a weaker antibody response compared to natural infection, a lower anti-Ptx IgG cutoff was used not to miss vaccination events47. The validity of this approach was tested on sera from individuals seeking medical care with confirmed pertussis infection (see Supplementary Methods).

In addition, a (cross-sectional) estimation of infection prevalence was conducted with an additional sample from the last PICO round (October 2024, n = 761) and compared with the longitudinal data from November 2022 and October 2024 and two cross-sectional serosurveys that were carried out in the past using the same measurements (Pienter-2, 2006/2007 and Pienter-3, 2016/2017)7,48. As the earlier surveys did not include sequential sampling, the definition used in our analysis so far could not be used for this comparison. As applied previously, we used anti-Ptx IgG concentrations of ≥100 IU/mL as the most stringent diagnostic criterium associated with a recent infection in the past 6 months44, concentrations between 50 and 100 IU/mL for an infection in the past 6–12 months, and those under 50 IU/mL for a less recent infection7,49.

Statistical analyses
Data cleaning and statistical analyses were performed using Rstudio (R version 4.4.0, R Core Team, Vienna, Austria) using packages nlme50, MatchIt51, effects52 and pROC53 and SAS v.9.4. (SAS Institute Inc., USA). For all analyses, age at the moment of inclusion (November 2022) was used, unless specified otherwise. The antibody data were log-transformed, and Loess (locally weighted scatterplot smoothing) curves were generated to visualize antibody concentrations across all ages at the start and end point of the study. A two-sided paired t-test on log-transformed data was used to compare the anti-Ptx IgG concentrations at the start and end point of the study for different age groups. An two-sided unpaired t-test on log-transformed data was used to compare the anti-Ptx IgG concentrations at the start of the study period between age groups.

Pertussis incidence per study round, cumulative incidence across the study period and seroprevalence estimated (with 95% confidence intervals (CI)) were calculated taking into account the survey design (with regions as strata and municipalities as clusters, where applicable), and weighted on age, sex, ethnic background and degree of urbanization following census data from the statistics Netherlands of the respected periods to match the general Dutch population, as described previously40. Estimates were provided for the overall population and by age group. For the pertussis incidence per study round, age during the specific round was used. Differences in seroprevalence between groups were assessed by estimating the parameters of the beta distribution using the method of moments. Risk ratios and their 95% Cis and two-sided p-values were calculated based on Monte Carlo simulations, and reported where applicable.

Waning of anti-Ptx IgG concentrations per age group was evaluated in individuals who remained uninfected (or up the point of infection) and unvaccinated with a linear mixed-effects model on log-transformed values, using the nlme package. The model contained random intercepts and slopes per age group, and the fixed effect was constructed as the interaction of time and age. The results are expressed as the predicted geometric mean concentrations of anti-Ptx IgG per age group and PICO study round with corresponding 95% CIs.

Pre-infection IgG and IgA antibody concentrations against B. pertussis antigens implicated in host colonization (FHA and Prn) were compared between participants with a confirmed infection (n = 38) and uninfected controls (n = 68) matched by age and sampling round. Antibodies to the independent antigen rubella served as control to monitor antibody variations. Log-transformed IgG concentrations were compared with a t-test and IgA concentrations with a Mann–Whitney U test due to not normally distributed data. Finally, to approximate the sensitivity and specificity of a potential correlate of protection, the Youden index of the receiver operating characteristics (ROC) curve analysis were used.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Outbreaks of pertussis are observed in many countries after the COVID-19 pandemic. In Europe, the highest incidence is reported in adolescents aged 10 to 19 years. Although multiple reasons are proposed to explain the outbreaks, the exact mechanisms are not known. This study included serum samples collected from 418 participants aged 2 to 87 years at five time points (November 2022-October 2024) of a nationwide prospective serosurveillance study conducted in the Netherlands and analyzed the concentrations of anti-PT IgG antibodies. They found that cumulative infection incidence was 6.3% in the Dutch population, and 35% in 6-18y-olds. The study provided an important information why the incidence of pertussis was high in adolescents during the outbreaks after the pandemic. The study design is proper and the finding is novel. I have following concerns which should be addressed first.\n1. A table to describe the current vaccination program of pertussis is needed, this will help understand the waning vaccine-induced immunity.\n2. The PICO study included about 10,000 participants. However, only 419 were randomly selected for the study. What is the statistical justification for the selection? Based on the early European seroprevalence studies (Wehlin et al APIMS 2021 and Berbers et al 2021), the sample size is estimated to be 250 samples/age group of 10 years e.g. 250 samples in age group of 20-29 years old.\nThis is especially important issue since the study failed to identify the cases with recent infections in age groups older than 19 years.\n3. The finding that participants of 6 to 18 years had low FHA IgG concentrations prior to infection was interesting. This finding was consistent with an early study conducted in Finland (He et al, JID 1994) in which they showed schoolchildren with high FHA IgG antibodies are protected during outbreaks.\n4. Another issue which should be discussed is that the FHA IgG antibodies can be induced by other Bordetella species or bacteria other than Bordetella.", "reviewer_2": "Globally, following the SARS CoV2 pandemic, there has been an increase in B. pertussis incidence. This manuscript contributes well to the literature related to B. pertussis immunology and antibody dynamics following the pandemic. This is also a good study design – following the same individuals overtime.\nOverall, beautiful figures  . Potentially add a : or . after each figure to separate the figure number and title.\nPlease review entire document and format – lots of mistakes picked up regarding font size, font type, in-text reference formatting etc.\nLine 29 – For Figure 1 – references 1-3 are added but within line 3, for the same figure only reference 2 has been added. Is there a reason for this?\nLine 32: The number 2 ends this sentence. I am assuming this is supposed to be reference 2 so it should be (2). Please correct.\nLine 46-48: The switch from the WCV to the ACV was made in 2005 – are the vaccine antigens for this vaccine the same as the vaccine that was given as a booster for the 4 year olds?\nLine 58: The age of the participants ranged from 2-87 years. Is this the minimum and maximum age for the enrolled participants or was this age range pre-determined? If pre-determined, how did you choose 2-87 years – I do see that this is mentioned in the limitations of the paper, however, potentially add a sentence describing the age in the methods so the reader quickly sees that this is the enrolled population.\nLine 61: Please correct formatting for this sentence – incorrect spacing between words.\nAre vaccines generally well received in the Netherlands? Any issues with vaccine hesitancy following the pandemic? Any vaccine coverage data available that could be added to the introduction – the vaccine schedule can also be added if word count allows?\nLine 69: You could remove the line : “The study has been described before (8-10)” and add it at the end of line 66 “….. until the end of 2024 (in total 13 study rounds), as previously described (8-10)”.\nI am assuming that the children <2 were not included in the study due to increased antibody levels in this age group due to vaccination?\nLine 115-118 – check font size and style – seems to have changed from preceding lines. Good practise is to check the entire document to ensure font size, font style, line spacing etc are all uniform.\nLine 124: How is incidence reported? This is also not clear in results e.g. x person-year or 100 000 population etc? Only the number is reported. The current way of reporting indicates that a prevalence is being presented instead of incidence as it’s a percentage that is reported.\nLine 132: 95% Cis – I am assuming this is confidence intervals – please correct to CI as abbreviated in line 125.\nLine 140: Formatting – italicize B. pertussis\nLine 185-188 – what other symptoms were reported? Flu like symptoms? Any pertussis specific symptoms? Can add a sentence or two to describe this.\nLines 218-226 – This is an important result as these data are lacking in the literature. If available to add in the time for waning? Instead of saying fastest antibody decay (line 223), add in an average time in months as well (if data is available). Some recent data from a longitudinal study from South Africa also showed rapid antibody waning (Moosa F, Kleynhans J, Makhathini L, du Plessis M, Tempia S et al). Bordetella pertussis infection and antibody dynamics in household cohorts in two South African communities, 2016 - 2018: findings from the PHIRST study. J Infect. 2025 Aug;91(2):106550. doi: 10.1016/j.jinf.2025.106550.\nLine 238 – “This data suggest a possible protective role ….” – this is a discussion point and not a result. Move this to discussion.\nLine 326 – I am assuming the number 3 at the end of the sentence is a reference – please format correctly.", "reviewer_3": "The authors describe immunity of pertussis during COVID-19 focusing on periods of low and high activity. The paper is flawed by the inability to distinguish between infection vs vaccine induced antibodies and using rather arbitrary cut offs for immunity against tetanus (>5 units) and categorization of infection against pertussis (lines 112-115). The authors should have used a consistent definition for both pertussis and tetanus (e.g. 2 or more fold change , 4 or more fold change).\nMore detailed feedback and comments.\nThe manuscript addresses an important and timely issue: the resurgence of Bordetella pertussis in the post–COVID-19 era. Pertussis remains the least controlled vaccine-preventable disease, and its re-emergence is a global concern. Countries that transitioned from whole-cell (wP) to acellular pertussis (aP) vaccines have consistently reported shorter-lived immunity, increased susceptibility in adolescents and adults, and recurrent epidemics every 3–5 years. The COVID-19 pandemic, with its widespread non-pharmaceutical interventions (NPIs), provided an unusual natural experiment by temporarily interrupting pathogen circulation. This interruption raised concerns that reduced natural immune boosting, combined with the already known rapid waning of aP-induced immunity, could exacerbate pertussis resurgence once restrictions were lifted. The manuscript correctly identifies this epidemiological and immunological interplay as the central concerns.\nThe authors address this issue by leveraging a nationwide prospective serosurveillance cohort to monitor antibody dynamics and infection incidence across age groups. Their data provide evidence that waning population immunity left certain groups (notably children and adolescents) more susceptible, which contributed to the sharp increase in cases from 2023 onwards. However, while the cohort design is robust and the longitudinal sampling a strength, the study is limited in its ability to fully disentangle the interplay between vaccination history, natural boosting, and other epidemiological drivers (e.g., bacterial adaptation, changes in diagnostic practices, or underdiagnosis).\nAnother limitation is the heavy reliance on serological markers (IgG, IgA against Ptx, FHA, and Prn) as analogs of immunity or to propose protection surrogates. While informative, antibody concentrations alone may not fully capture protective immunity, especially given the growing recognition of the role of cellular responses in pertussis control. The authors acknowledge waning immunity but could have more critically contextualized their findings within the broader debate over the adequacy of current pertussis vaccination strategies.\nIn summary, the manuscript provides valuable national data on pertussis resurgence after the COVID-19 pandemic and highlights waning vaccine-induced immunity as a major driver. Yet, its framing could be strengthened by situating the findings within the broader global context of pertussis resurgence, addressing alternative explanations beyond antibody waning, and more explicitly discussing implications for vaccination policy.\nSuggested corrections\nMajor\n1. From line 39, the authors state that pertussis is often accompanied by whooping cough, but this is characteristic of the paroxysmal stage. In the catarrhal phase, symptoms are nonspecific and resemble those of a common cold. To avoid misinterpretation, the reviewer suggests deleting the second sentence of this paragraph and instead clarifying that during the paroxysmal phase—the most severe and symptomatic stage—patients experience episodes of spasmodic coughing fits, inspiratory whooping, and post-tussive vomiting.\n2. The authors repeatedly refer to the use of tetanus toxin in their experiments. However, due to biosafety constraints, toxoids are more commonly used in these contexts. Could the authors clarify whether the toxic or atoxic form of the protein was employed?\n3. While the Dutch National Immunization Programme (NIP) is publicly available, the reviewer recommends briefly outlining it in the introduction to aid reader understanding. To my understanding, pertussis acellular components are currently included in the hexavalent vaccine administered at 2/3, 5, and 12 months of age, with a booster at 5 years (trivalent vaccine). No further boosters are given, except in pregnant women to protect newborns early in life.\n4. It is unclear why the anti-rubella response was used to assess vaccination history, since rubella vaccination is given separately and not concomitantly with pertussis or tetanus toxoid. In the Netherlands, MMR is administered at 14 months and 3 years of age, which differs from the pertussis vaccination schedule. If the aim was to assess recent pertussis vaccination, a non-vaccine pertussis antigen such as adenylate cyclase toxin might have been more appropriate. Could the authors explain the rationale behind selecting anti-rubella antibodies as a control? This is insufficiently clarified in the text and would benefit from further explanation.\n5. In Figure 6, the authors should visually separate the anti-pertussis proteins results from those of anti-rubella antigens. The reviewer recommends adding a vertical, thick dotted or dashed line running top-to-bottom after panels C and F.\n6. Although the manuscript mentions that wP vaccination (and natural infection) primarily induces Th1/Th17 responses—contrasting with the Th2 bias of aP vaccines—the authors did not examine the IgG1/IgG2 ratio in sera from convalescent, recently immunized, and low-titer individuals. This omission is a limitation, since IgG2 is suboptimal for opsonization and phagocytosis, and absolute quantification of aP-induced antibodies may overlook this. This point should be discussed in the manuscript. Moreover, recent studies suggest that aP vaccines generate antigen-specific regulatory T cells that compromise the induction of long-term mucosal immunity in the respiratory tract.\n7. Line 358: Please clarify what the rubella material supplied by GenWay comprises. Is it a surface antigen or an inactivated virus?\nMinor\n1. Some acronyms should be standardized. For instance, Ptx IgG appears interchangeably as “Ptx IgG,” “IgG Ptx,” and “anti-Ptx IgG.” The reviewer recommends consistently using the last form.\n2. line 133: “CIs.”\n3. Line 87: should read anti-tetanus toxoid, since the administered form is the anatoxin.\n4. Line 141: Bordetella pertussis should be italicized. Line 154: i.e. (as a Latin abbreviation) should also be italicized.\n5. Line 153: should read 0.1 IU/mL.\n6. Line 170: should say “dashed lines” instead of “dotted lines.”\n7. Line 195: sentence structure is awkward and should be revised for clarity.\n8. Figure 3B: the double line of numbers under the x-axis is visually unappealing. The reviewer recommends keeping only the even numbers (upper line), as it is clear that odd ages are included.\n9. Lines 238–239: the measurement units are missing after “1.0” and “2.1.”\n10. Line 298: the comma after “outbreaks” should be removed.\n11. Line 314: should read CD4+ T.\n12. Line 356: Pertactin (or pertactin, as the autors prefer) P.69\n13. Line 360: should read “or anti-human IgA.”\n14. Line 363: should read geometric mean.\n15. Line 364: should read mean fluorescence intensity (MFI).\n16. Line 366: delete the comma after “infection”.\n17. Line 368: sentence should be in past tense.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Thank you for giving us the opportunity to revise our manuscript. We appreciate the reviewers’ comments and suggestions. We have carefully addressed all points raised by the reviewers and provided detailed responses to each comment below. When necessary, we have made changes to the manuscript to strengthen our study. All changes in the manuscript are shown using track changes, and the corresponding line number are included in our responses. We hope that our revisions and responses meet your expectations, and we look forward to your feedback.\nReviewer #1 (Remarks to the Author):\nOutbreaks of pertussis are observed in many countries after the COVID-19 pandemic. In Europe, the highest\nincidence is reported in adolescents aged 10 to 19 years. Although multiple reasons are proposed to explain\nthe outbreaks, the exact mechanisms are not known. This study included serum samples collected from 418\nparticipants aged 2 to 87 years at five time points (November 2022-October 2024) of a nationwide prospective\nserosurveillance study conducted in the Netherlands and analyzed the concentrations of anti-PT IgG\nantibodies. They found that cumulative infection incidence was 6.3% in the Dutch population, and 35% in 6-\n18y-olds. The study provided an important information why the incidence of pertussis was high in adolescents\nduring the outbreaks after the pandemic. The study design is proper and the finding is novel. I have following\nconcerns which should be addressed first.\n1. A table to describe the current vaccination program of pertussis is needed, this will help understand the\nwaning vaccine-induced immunity.\nResponse:\nWe agree that the pertussis vaccination timeline is a determining factor in waning immunity so we added a\ntable in the Methods addressing this, including the most relevant changes.\n2. The PICO study included about 10,000 participants. However, only 419 were randomly selected for the study.\nWhat is the statistical justification for the selection? Based on the early European seroprevalence studies\n(Wehlin et al APIMS 2021 and Berbers et al 2021), the sample size is estimated to be 250 samples/age group of\n10 years e.g. 250 samples in age group of 20-29 years old.\nThis is especially important issue since the study failed to identify the cases with recent infections in age groups\nolder than 19 years.\nResponse: Thank you for your comments regarding the participant selection and sample size in the PICO study.\nTo clarify, while the PICO study included approximately 10,000 unique participants across multiple rounds, a\nmuch smaller selection of participants was available providing serum samples at multiple timepoints. During\nthe 9th, 10th and 11th sample round, about 4000 individuals were participating in the study, of which 92% was\n>18 years.\nThe studies mentioned by the reviewer used a cross-sectional design with an expected 20% prevalence which is\nnot comparable to our study. Our study has a prospective design, in which we can determine infection at\nindividual basis by analyzing changes in antibody concentration between timepoints instead of measuring\nseroprevalence cross-sectionally, which was previously shown to demonstrate a cross-sectional seroprevalence\nof 5.9% (1).\nTo detect an overall incidence of around 6% with a one-sided precision of 2.5%, 80% statistical power and an\nalpha of 5%, we calculated a minimum number of 347 individuals. Taking into account a potential 15% loss of\nfollow-up samples, at least 408 participants were needed in the longitudinal analysis.\nBecause incidence rates are expected to be different between age groups, the precision varies. This was\naccounted for by taking narrower age ranges in the younger age groups were a higher incidence is expected\n(1). Precision by incidence for our study are as follows:\n- 2-5 years age group: a 6% incidence rate with a precision of 5.5%.\n- 6-18 years age group: a 12% incidence with a precision of 6%\n- 19-59 years age group: a 5% incidence with a precision of 4%. However, in the final manuscript, we\nreported the 19-39 and 40-59 year age groups separately.\n- 60 years and older: a 6% incidence, with a precision of 5.5%.\nTaking into account a potential 15% loss of follow-up samples in each age group, we aimed to include at least\n84 individuals in the 2-5 years age group, 133 individuals in the 6-18 years age group, 134 in the 19-59 years\nage group and 84 in the 60+ age group. The desired sample size was not available for the 2–5 year age group;\ntherefore, all 43 available individuals were included.\nIn our study we focused on the overall incidence in the Dutch population, and zoomed in on the incidence in\nchildren aged 6-18 years. A 6.3% overall incidence could be determined with 2.3% precision. In children aged 6-\n18 years, a 35.4% incidence could be determined with 8.0% precision, which shows that we have an accurate\nincidence estimation in these age groups.\nThe incidence rate of the 19-39, 40-59 and 60+ year age groups appeared lower than the overall 5-6% we\nexpected. Therefore we performed an additional cross-sectional seroprevalence analysis in the last sampling\nround. This approach indeed resulted in the identification of infected cases among individuals aged 40-59\nyears, pointing to an underestimation of the number of pertussis infections in this age group. We hope this\nmeets the major point addressed by the reviewer.\nSince the justification for the statistical power of the participant numbers included in our study was lacking in\nour initial submission, we added a paragraph in the Methods section:\nLine 84-88: For the overall analysis, we expected a 6% incidence rate (7) and aimed for a one-sided precision of 2.5%,\nresulting in a minimum required sample size of 347 individuals (α = 0.05, power = 80%). Taking into account a potential loss\nof 15% of follow-up samples, at least 408 participants were needed in the longitudinal analysis. For all age groups we were\nable to include sufficient individuals, except for the youngest age group (2-5 years), where only 43 individuals were available\nwho were all included.\n3. The finding that participants of 6 to 18 years had low FHA IgG concentrations prior to infection was\ninteresting. This finding was consistent with an early study conducted in Finland (He et al, JID 1994) in which\nthey showed schoolchildren with high FHA IgG antibodies are protected during outbreaks.\nResponse: We thank the reviewer for pointing to an earlier study that the other way around, high antibody\nlevels for FHA correlated with protection . We have added this the to the discussion, including the citation.\nLine 317-318: This finding aligns with a previous study by He et al. conducted in Finland, demonstrating that high anti-FHA\nIgG antibodies are protective in schoolchildren during outbreaks (2).\n4. Another issue which should be discussed is that the FHA IgG antibodies can be induced by other Bordetella\nspecies or bacteria other than Bordetella.\nResponse: We agree with the reviewer that anti-FHA antibodies can be induced by other species as well. We\nhave added a sentence in the discussion to address this point.\nLine 326-330: However, anti-FHA antibodies are not only induced by B. pertussis infection and vaccination, but also by other\nspecies such as Bordetella parapertussis (24). Therefore, increased anti-FHA IgG concentration does not necessarily indicate\nprior exposure to B. pertussis, but the exposure to B. parapertussis may also contributed to increased anti-FHA IgG\nconcentrations.\nReviewer #2 (Remarks to the Author):\nGlobally, following the SARS CoV2 pandemic, there has been an increase in B. pertussis incidence. This\nmanuscript contributes well to the literature related to B. pertussis immunology and antibody dynamics\nfollowing the pandemic. This is also a good study design – following the same individuals overtime.\nOverall, beautiful figures . Potentially add a : or . after each figure to separate the figure number and title.\nPlease review entire document and format – lots of mistakes picked up regarding font size, font type, in-text\nreference formatting etc.\nResponse:\nThank you for your appreciation. We thoroughly went through the manuscript again to apply consistent\nformatting, including appropriate separators.\nLine 29 – For Figure 1 – references 1-3 are added but within line 3, for the same figure only reference 2 has\nbeen added. Is there a reason for this?\nResponse:\nThe references 1-3 support the statement made in lines 27-29. These references show the impact of COVID-19\nNPIs on the incidence of vaccine preventable diseases, among which pertussis, in The Netherlands at the start\nof the pandemic (1). This study shows the reduction in vaccine preventable diseases incidence in the first year\nof the COVID-19 pandemic. The second reference shows the RIVM report of the surveillance of the National\nImmunisation Programme in the Netherlands in 2023-2024 which included the number of notifications of B.\npertussis before, during and after the COVID-19 pandemic (2). The third reference is an assessment of ECDC\nand shows limited circulation of pertussis in the EU during the COVID-19 pandemic and the increase in 2023\nand 2024 in multiple European countries (3).\nWhile reference number 1 and 3 refers to the general impact of COVID-19 NPIs (1), and assessments from ECDC\n(3), reference number 2 in lines 32-33 refers to the increase in pertussis notifications specifically related to\nNetherlands, which we have specifically added now to the sentence. Hope this clarifies the confusion.\nLine 32-33: Since May 2023, pertussis notifications rose, cumulating in the largest pertussis outbreak to date since 2012 in\nthe Netherlands (Figure 1) (2).\nLine 32: The number 2 ends this sentence. I am assuming this is supposed to be reference 2 so it should be (2).\nPlease correct.\nResponse: We do not observe these formatting irregularities in our original submission files and the pdf\ndownloaded following the initial submission. However, to minimize chances of errors introduced on our end,\nwe thoroughly went through the manuscript again for consistent formatting to avoid errors.\nLine 46-48: The switch from the WCV to the ACV was made in 2005 – are the vaccine antigens for this vaccine\nthe same as the vaccine that was given as a booster for the 4 year olds?\nResponse:\nPrincipally yes. ACV given as primary vaccine since 2005 was the same as the one given as a booster for the 4\nyear olds. Since 2017 booster vaccination is? still conducted with the same ACV, but with reduced\nconcentrations of Bp components. This has been stated In the Methods section (lines 114-115). To clarify this\nfurther, we added a table in the Methods section describing the pertussis vaccination schedule in the\nNetherlands in which we highlighted most relevant changes.\nLine 58: The age of the participants ranged from 2-87 years. Is this the minimum and maximum age for the\nenrolled participants or was this age range pre-determined? If pre-determined, how did you choose 2-87 years\n– I do see that this is mentioned in the limitations of the paper, however, potentially add a sentence describing\nthe age in the methods so the reader quickly sees that this is the enrolled population.\nResponse:\nThe PICO study started with participants from the PIENTER-3 study which was conducted in 2016/2017. In the\nsecond (June 2020) and sixth (November 2021) sampling rounds, additional individuals were included, with\npredominately younger and older age groups due to higher drop-outs rates (4). Individuals needed to be at\nleast 1 year of age to be eligible to participate in the PICO study. Since our study including participants from\none year after the last upsampling in November 2022, the youngest age in our study population was 2 years.\nWe randomly included participants per age group, which resulted in our oldest individual to be 87 years of age.\nThe reviewer is right that this age range is lacking, so we added this in the methods section accordingly.\nLine 81-82: Here, we randomly selected an age-stratified subset with completed questionnaires and samples (n=419) from\nstudy rounds 9–13 (November 2022-October 2024) (age range: 2-87 years).\nLine 61: Please correct formatting for this sentence – incorrect spacing between words.\nResponse: We do not observe these formatting irregularities in our original submission files and the pdf\ndownloaded following the initial submission. However, to minimize chances of errors introduced on our end,\nwe thoroughly went through the manuscript again for consistent formatting to avoid errors.\nAre vaccines generally well received in the Netherlands? Any issues with vaccine hesitancy following the\npandemic? Any vaccine coverage data available that could be added to the introduction – the vaccine schedule\ncan also be added if word count allows?\nResponse:\nThank you for your comment. Vaccines are generally well received in The Netherlands, but a slight downward\ntrend of vaccination coverage in the Netherlands has indeed been observed but became already eminent prior\nto the pandemic (5). Of the infants born in 2019, 92.2% have received all DKTP vaccinations at 2 years of age.\nTo what extend the COVID-19 pandemic further contributed to increased vaccine hesitancy is currently being\ninvestigated, but seems not to have affected DKT vaccination not a large extend.\nWe added a table in the methods indicating the pertussis vaccination schedule in the Netherlands and highlight\nrecent and most relevant changes.\nWe also added a sentences about decreasing vaccination coverage in the introduction:\nLine 53-56: Of special concern is the decrease in vaccination coverage in the Netherlands that has been observed over the\npast years, with 92.2% of the infants born in 2019 who have been fully vaccinated by the age of two (8).\nLine 69: You could remove the line : “The study has been described before (8-10)” and add it at the end of line\n66 “….. until the end of 2024 (in total 13 study rounds), as previously described (8-10)”.\nResponse: Changed accordingly\nI am assuming that the children <2 were not included in the study due to increased antibody levels in this age\ngroup due to vaccination?\nResponse: Children <2 years are not included due to inclusion criteria of the PICO study. More details are\nprovided in the response to the remark of the age of the participants included in the study.\nLine 115-118 – check font size and style – seems to have changed from preceding lines. Good practise is to\ncheck the entire document to ensure font size, font style, line spacing etc are all uniform.\nResponse: We do not see these formatting irregularities in our original submission files and the pdf\ndownloaded following the initial submission. However, to minimize chances of errors introduced on our end,\nwe check the revised submission files for consistent formatting to avoid errors.\nLine 124: How is incidence reported? This is also not clear in results e.g. x person-year or 100 000 population\netc? Only the number is reported. The current way of reporting indicates that a prevalence is being presented\ninstead of incidence as it’s a percentage that is reported.\nResponse:\nThe incidence is reported as the percentage of the Dutch population that was infected with pertussis over a\ntwo-year period. The calculation of the incidence is described in the methods (Line 150-154). Because this was\nan outbreak instead of a regular season, we feel that it was more appropriate to report the incidence over the\nentire study period encompassing the whole outbreak. To clarify this in the results, we additionally stated that\nthe incidence is reported over a two-year period:\nLine 210-212: The weighted cumulative incidence in the Dutch population (2-87 years) during this two-year period was\nestimated at 6.3%. The highest incidence (35.4%) was found among age group 6-18 years (Figure 3B, Table 2).\nLine 132: 95% Cis – I am assuming this is confidence intervals – please correct to CI as abbreviated in line 125.\nResponse: Correct, 95% Cis is changed to 95% CIs\nLine 140: Formatting – italicize B. pertussis\nResponse: Changed\nLine 185-188 – what other symptoms were reported? Flu like symptoms? Any pertussis specific symptoms?\nCan\nadd a sentence or two to describe this.\nResponse:\nWe agree that it is important to clarify the type of symptoms that were reported.\nIn the questionnaire, participants filled in whether they had any COVID-19 related symptoms since the previous\nsampling moment. The symptoms include general symptoms such as fever and headache, respiratory\nsymptoms like cough, sore throat and runny nose and gastrointestinal symptoms like diarrhea (6). Because of\nthe rise in pertussis notifications in the last sampling round (PICO13), a specific question was added about\nprolonged coughing (>2 weeks).\nTo clarify the type of symptoms that were reported, we added the following sentence to the methods section:\nLine 100-102: Each round participants were requested to report respiratory symptoms including cough, sore throat and\nrunny nose and fever which are associated with B. pertussis infection (7).\nLine 215-216: Individuals with serological evidence of B. pertussis infection did not report increased symptoms compared\nto uninfected individuals\nLines 218-226 – This is an important result as these data are lacking in the literature. If available to add in the\ntime for waning? Instead of saying fastest antibody decay (line 223), add in an average time in months as well\n(if data is available). Some recent data from a longitudinal study from South Africa also showed rapid antibody\nwaning (Moosa F, Kleynhans J, Makhathini L, du Plessis M, Tempia S et al). Bordetella pertussis infection and\nantibody dynamics in household cohorts in two South African communities, 2016 - 2018: findings from the\nPHIRST study. J Infect. 2025 Aug;91(2):106550. doi: 10.1016/j.jinf.2025.106550.\nResponse: Thank you for highlighting the importance of antibody waning. We appreciate the suggestion to\nprovide an average waning. Under the assumption that waning is linear and stable over time, we added the\nduration of antibody decay to 50% for the four different age groups.\nIn the results section we now report the starting concentrations as well as decay rate for the different age\ngroups:\nLine 256-264: The 2-5 years-olds showed the highest anti-Ptx IgG concentrations at the start of the study (16.45 IU/mL;\np<0.05 compared to other age groups). The 6-12 years age group displayed lower antibody concentrations at the start of\nthe study period compared to adults (5.83 IU/mL vs 7.28 IU/mL; p=0=0.009). The fastest antibody decay was observed in the\n2-5 years age group with the duration of antibody decay to 50% of 17.8 months (Figure 5; p<0.001 for the slope). Antibody\ndecay was also noticed in the 6-12, 13-18 and ≥19 years age group (p<0.001 for the slopes). The duration of antibody decay\nto 50% in the 6-12, 13-18 and ≥19 years age group was 48.5, 102.5 and 84.1 months respectively.\nLine 238 – “This data suggest a possible protective role ….” – this is a discussion point and not a result. Move\nthis to discussion.\nResponse: This sentence is removed from the results\nLine 326 – I am assuming the number 3 at the end of the sentence is a reference – please format correctly.\nResponse: We do not see these formatting irregularities in our original submission files and the pdf\ndownloaded following the initial submission. However, to minimize chances of errors introduced on our end,\nwe check the revised submission files for consistent formatting to avoid errors.\nReviewer #3 (Remarks to the Author):\nThe authors describe immunity of pertussis during COVID-19 focusing on periods of low and high activity. The\npaper is flawed by the inability to distinguish between infection vs vaccine induced antibodies and using rather\narbitrary cut offs for immunity against tetanus (>5 units) and categorization of infection against pertussis\n(lines 112-115). The authors should have used a consistent definition for both pertussis and tetanus (e.g. 2 or more\nfold change , 4 or more fold change).\nResponse:\nThe reviewer raises two important issues: the discrimination between infection and vaccine-induced\nantibodies, and the use of cutoffs.\nThe first issue of discriminating between infection and vaccine-induced antibodies is fundamental to our\nstudies, where we aimed to identify infections and not erroneously classify vaccination events as infections.\nHere we disagree with the statement made by the reviewer that we cannot distinguish between infection and\nvaccine-induced antibodies, as we paid close attention to, as accurately as possible, classify individuals with\nincreased anti-PT IgG concentrations as being recently infected. Indeed, the detection of antibodies to\npertussis itself cannot discriminate between infection and vaccination. This is why we added a validated\nantibody assay for tetanus toxin to reliably detect instances of vaccination as this is part of the pertussis\nvaccine as well which is a correct approach. We do realize however, that no laboratory test is 100% accurate\nand that still some misclassification can occur. This we acknowledge in the Discussion of our manuscript lines\n389-391.\nDifferent cut-offs were applied for longitudinal respectively cross-sectional data analyses because this results in\nmore accurate (specificity and sensitivity) identification of infections. By doing so, we could benefit from the\nlongitudinal sampling to identify infections during the study period (a ≥2-fold increase together with a final\nconcentrations of ≥20 IU/mL). Whilst fold-changes is a sensitive method to identify infections, this is less\naccurate at low concentrations due to more background variation, which is why we applied a concentration-\nbased cutoff to avoid cases being falsely classified as infections (20 IU/mL for anti-Ptx IgG). As the antibody\nresponse to vaccination is weaker compared to infection, we decreased the cut-off for anti-Ptx-IgG from 20\nIU/mL to 5 IU/mL. This lower cut-off revealed four additional vaccinated individuals who otherwise would have\nbeen missed. To clarify why we used a different cut-off in longitudinal samples, we changed the following\nsentence to the methods section:\nLine 122-125: Because the Tdap booster vaccination contains reduced concentrations of the pertussis components, and\nvaccination induces a weaker antibody response compared to natural infection, a lower anti-Ptx IgG cutoff was used to not\nmiss vaccination events.\nFor the additional cross-sectional sampling, we could only apply absolute levels indicative for recent infection\nto allow comparison between studies (<50 IU/mL, 50-100 IU/mL and ≥100 IU/mL). The cut-off of 100 IU/mL is\nalso used in the diagnostic lab as cut-off for recent infection, while the 50-100 IU/mL cutoff is used by multiple\nstudies (1, 8).\nThese different analyses have been described separately in our Methods section and these cutoffs have been\napplied consistently (lines 112-126).\nMore detailed feedback and comments.\nThe manuscript addresses an important and timely issue: the resurgence of Bordetella pertussis in the post–\nCOVID-19 era. Pertussis remains the least controlled vaccine-preventable disease, and its re-emergence is a\nglobal concern. Countries that transitioned from whole-cell (wP) to acellular pertussis (aP) vaccines have\nconsistently reported shorter-lived immunity, increased susceptibility in adolescents and adults, and recurrent\nepidemics every 3–5 years. The COVID-19 pandemic, with its widespread non-pharmaceutical interventions\n(NPIs), provided an unusual natural experiment by temporarily interrupting pathogen circulation. This\ninterruption raised concerns that reduced natural immune boosting, combined with the already known rapid\nwaning of aP-induced immunity, could exacerbate pertussis resurgence once restrictions were lifted. The\nmanuscript correctly identifies this epidemiological and immunological interplay as the central concerns.\nThe authors address this issue by leveraging a nationwide prospective serosurveillance cohort to monitor\nantibody dynamics and infection incidence across age groups. Their data provide evidence that waning\npopulation immunity left certain groups (notably children and adolescents) more susceptible, which\ncontributed to the sharp increase in cases from 2023 onwards. However, while the cohort design is robust and\nthe longitudinal sampling a strength, the study is limited in its ability to fully disentangle the interplay between\nvaccination history, natural boosting, and other epidemiological drivers (e.g., bacterial adaptation, changes in\ndiagnostic practices, or underdiagnosis).\nResponse:\nThank you for your constructive feedback. We agree that disentangling the complex interplay between the\nvarious factors is important, and do acknowledge that our study does not, and cannot fully separate this\ncomplex interplay. This would require a prospective study design that follows the same individuals from the\ninfant immunization schedule all the way into adolescence to generate a complete history of vaccination and\nexposures to the bacterium, paired with tracking the bacteria circulating during that study period and\nperforming molecular typing of all infections. Our study was restricted to antibody response data only, limited\nto infection data from current reporting systems, which we addressed in our discussion.\nHowever, the serosurveillance approach allows the detection of pertussis infections not driven by clinical\ncriteria nor by clinical reporting systems, which is anticipated to identify a much higher B. pertussis infection\nprevalency, mainly among those where B. pertussis infections have low clinical impact. This is indeed what we\ndemonstrated among the different age groups. In our study, we detected a higher percentage of the population\nto be infected with B. pertussis during the peak in notifications. This suggest much higher circulation of B.\npertussis compared to that of the clinical notifications, which we highlighted in our discussion with the\nfollowing sentence:\nLine 331-337: The increased number of infections in the 6-18 year age group was also evident in the notification data of\nsyndromic cases in the Netherlands and other countries (2, 3, 25). However, according to our serological criteria of infection,\nwe detected a much higher infection rate (35.4%) compared to the number of notifications in the 6-18 years age group\n(0.24%). This difference is likely caused by the high prevalence of mild and asymptomatic infections not leading to seeking\nmedical care, against a relative low clinical manifestation of B. pertussis infections within this age group in general (5, 12,\n26).\nDespite the use of serological tools, we were able to detect vaccination event based on combined anti-Ptx and\nanti-Ttx IgG boosts, as described in the previous comment. The reviewer is correct that we have do not have\nvaccination records on individual basis. Population-wide the vaccination coverage is slightly decreasing. To\nclarify this to the reader, a sentence in the introduction regarding vaccination coverage based on the Dutch\nvaccination coverage report was added:\nLine 53-59: Of special concern is the decrease in vaccination coverage in the Netherlands that has been observed over the\npast years, with 92.2% of the infants born in 2019 who have been fully vaccinated by the age of two (8). Since 2019, the\nmaternal Diphtheria-Tetanus-acellular Pertussis (Tdap) vaccination is implemented in the Dutch NIP to increase the level of\nmaternally-derived immunological protection in the first months post birth (2). However, this is not implemented globally\nand pertussis remains one of the least controlled vaccine preventable diseases.\nBacterial adaptation is an important factor in the increased incidence of pertussis infections, especially in\ncombination with new vaccine schedules. We know that after the COVID-19 pandemic, fewer pertactin\ndeficient pertussis strain were circulating compared to the years before COVID-19 (9). We were not able to\ninclude this in in our study due to the nature of our study, but we have added a paragraph to the discussion to\naddress this point:\nLine 366-374: These findings show the need of improved pertussis vaccines that induce local antibodies and T-cell\nresponses. This is essential for prevention of colonization and clearance of B. pertussis and improve the control of pertussis\ncirculation. It is known that there is continuous adaptation of B. pertussis to its host (35). Since the introduction of the aP\nvaccine, an increase of Prn-deficient strains has been observed in many countries (36). However, typing of Dutch pertussis\nisolates in 2023 and 2024 showed less circulation of Prn-deficient pertussis strains, which we think could be related to the\ndecline in immunity reported in our study (37). This ongoing bacterial adaptation is an additional challenge to long-term\npertussis control and also shows the need for continued B. pertussis molecular surveillance, in addition to monitoring of\nimmunity in the population.\nAnother limitation is the heavy reliance on serological markers (IgG, IgA against Ptx, FHA, and Prn) as analogs of\nimmunity or to propose protection surrogates. While informative, antibody concentrations alone may not fully\ncapture protective immunity, especially given the growing recognition of the role of cellular responses in\npertussis control. The authors acknowledge waning immunity but could have more critically contextualized\ntheir findings within the broader debate over the adequacy of current pertussis vaccination strategies.\nIn summary, the manuscript provides valuable national data on pertussis resurgence after the COVID-19\npandemic and highlights waning vaccine-induced immunity as a major driver. Yet, its framing could be\nstrengthened by situating the findings within the broader global context of pertussis resurgence, addressing\nalternative explanations beyond antibody waning, and more explicitly discussing implications for vaccination\npolicy.\nResponse:\nWe agree that measuring antibody concentrations do not provide a complete picture of immunity against\npertussis, though earlier studies have already pointed out that antibodies against FHA correlate with protection\nagainst pertussis (2). Anti-FHA IgG concentrations plays an important role in prevention of colonization of\npertussis (10-12).\nIt would be certainly valid to study cellular immune profiles in our study cohort as long term protection is also\ndependent on cellular immune responses. Unfortunately, due to the nature of our study, we had no access to\ncellular samples. We know from previous research that aP vaccination has a suboptimal induction of a T-cell\nmemory response which is unable to prevent infection (13), which is also described in the discussion (Line 359-\n364).\nLine 364-365: Although we do present clear evidence of changes in humoral immunity during COVID-19, analyses of\nchanges in T cell immunity is warranted.\nWe thank the reviewer for pointing out that we could improve implications for vaccination policies. In the\ndiscussion we already stated that the national immunization program shifted the booster vaccination from 4 to\n5 years to enhance protection at older ages (Line 402-404). We provided an additional sentence in the\ndiscussion about the need of improved vaccines and have made several modifications as described above.\nLines 366-368: These findings show the need of improved pertussis vaccines that induce local antibodies and T-cell\nresponses. This is essential for prevention of colonization and clearance of B. pertussis and improve the control of pertussis\ncirculation.\nSuggested corrections\nMajor\n1. From line 39, the authors state that pertussis is often accompanied by whooping cough, but this is\ncharacteristic of the paroxysmal stage. In the catarrhal phase, symptoms are nonspecific and resemble those of\na common cold. To avoid misinterpretation, the reviewer suggests deleting the second sentence of this\nparagraph and instead clarifying that during the paroxysmal phase—the most severe and symptomatic stage—\npatients experience episodes of spasmodic coughing fits, inspiratory whooping, and post-tussive vomiting.\nResponse: The reviewer is correct that the symptom could be described more accurately. As mentioned in the\ncomment we changed this to:\nLine 41-45: Pertussis is a highly contagious respiratory infection. During the catarrhal stage, symptoms mostly resemble\nthose of a common cold. In the paroxysmal phase, which is the most symptomatic stage, symptoms are often coughing,\ninspiratory whooping and post-tussive vomiting (4). Infections can occur at any age but can cause serious disease in\n(predominantly unvaccinated) infants and older adults (4).\n2. The authors repeatedly refer to the use of tetanus toxin in their experiments. However, due to biosafety\nconstraints, toxoids are more commonly used in these contexts. Could the authors clarify whether the toxic or\natoxic form of the protein was employed?\nResponse: Thank you for your question. In our experiments, we used the toxic form of tetanus (tetanus toxin),\nwhich is immobilized to the beads once conjugated that subsequently are used through multiple experiments.\nAll procedures were carried out in accordance with institutional biosafety guidelines.\n3. While the Dutch National Immunization Programme (NIP) is publicly available, the reviewer recommends\nbriefly outlining it in the introduction to aid reader understanding. To my understanding, pertussis acellular\ncomponents are currently included in the hexavalent vaccine administered at 2/3, 5, and 12 months of age,\nwith a booster at 5 years (trivalent vaccine). No further boosters are given, except in pregnant women to\nprotect newborns early in life.\nResponse: We agree that the Dutch NIP should be described in the manuscript to aid the understanding of\nwaning immunity. We added a table in the methods section which described the pertussis vaccination schedule\nin the Netherlands and highlight recent and most important changes.\n4. It is unclear why the anti-rubella response was used to assess vaccination history, since rubella vaccination is\ngiven separately and not concomitantly with pertussis or tetanus toxoid. In the Netherlands, MMR is\nadministered at 14 months and 3 years of age, which differs from the pertussis vaccination schedule. If the aim\nwas to assess recent pertussis vaccination, a non-vaccine pertussis antigen such as adenylate cyclase toxin\nmight have been more appropriate. Could the authors explain the rationale behind selecting anti-rubella\nantibodies as a control? This is insufficiently clarified in the text and would benefit from further explanation.\nResponse:\nWe would like to point out that we did consider the anti-rubella response, but not to assess recent pertussis\nvaccination but as an independent assessment to monitor antibody variations over time between consecutive\nserum samples. Instead we used anti-tetanus toxin as the more accurate approach in this respect. We consider\nrubella antibodies to provide a stable pattern over time after vaccination, and because of absence of any\nrubella circulation during the study period.\nThe reviewer is right that this not clarified sufficiently in the manuscript. We changed to paragraphs in the\nmethods section to clarify this:\nLine 105-108: Serum samples were tested for anti-Ptx, anti-tetanus toxin (Ttx; to control for vaccination) and anti-rubella\n(as an independent control to monitor antibody variation) IgG antibodies at all sampling rounds using an in-house validated\nfluorescent bead-based multiplex immunoassay as described before.\nLine 167-168: Antibodies to the independent antigen Rubella served as control for to monitor antibody variations\n5. In Figure 6, the authors should visually separate the anti-pertussis proteins results from those of anti-rubella\nantigens. The reviewer recommends adding a vertical, thick dotted or dashed line running top-to-bottom after\npanels C and F.\nResponse: We agree that separation of the pertussis antigens and the rubella control improves the figure. We\nadded a the new figure to the manuscript.\n6. Although the manuscript mentions that wP vaccination (and natural infection) primarily induces Th1/Th17\nresponses—contrasting with the Th2 bias of aP vaccines—the authors did not examine the IgG1/IgG2 ratio in\nsera from convalescent, recently immunized, and low-titer individuals. This omission is a limitation, since IgG2\nis suboptimal for opsonization and phagocytosis, and absolute quantification of aP-induced antibodies may\noverlook this. This point should be discussed in the manuscript. Moreover, recent studies suggest that aP\nvaccines generate antigen-specific regulatory T cells that compromise the induction of long-term mucosal\nimmunity in the respiratory tract.\nResponse: Thank you for your comment. We agree that antibody subclass ratios could help for the complete\ninterpretation regarding the Th1/Th2 profile. For this Th2-biased response following aP immunization also IgG4\nis commonly used instead of IgG2. We added a paragraph to the discussion highlighting the differences in\nsubclass ratios.\nLine 360-364: This is also observed in antibody subclasses. After aP vaccination, a higher proportion of anti-Ptx IgG2 and\nIgG4 is found (32, 33). Compared to IgG1 and IgG3, IgG2 is less efficient at activating the complement system, while IgG4 is\nunable to activate the complement system, which limits the effectiveness in clearance of the infection (34).\n7. Line 358: Please clarify what the rubella material supplied by GenWay comprises. Is it a surface antigen or an\ninactivated virus?\nResponse: The rubella material we used in our assay is an inactivated virus (HPV-77 strain) provided by Aviva\nSystems Biology, which acquired Genway Biotech. We have changed this in the methods section.\nLine 412-415: The beads were conjugated to Ptx (The Native Antigen Company, Kidlington, UK, PT-TNF-100), FHA\n(Kaketsuken, Kumamoto, Japan), Pertactin P.69 (expressed and purified from an E. coli construct), Ttx (Sigma-Aldrich, St.\nLouis, MO, T3194), and an inactivated rubella virus HPV-77 (Aviva Systems Biology, San Diego, CA, GWB-HG4389).\nMinor\n1. Some acronyms should be standardized. For instance, Ptx IgG appears interchangeably as “Ptx IgG,” “IgG\nPtx,” and “anti-Ptx IgG.” The reviewer recommends consistently using the last form.\nResponse: The reviewer is right that a consistent form of anti-Ptx IgG should be used. We changed all forms to\nanti-Ptx IgG.\n2. line 133: “CIs.”\nResponse: Changed\n3. Line 87: should read anti-tetanus toxoid, since the administered form is the anatoxin.\nResponse: I our assay we used tetanus toxin to measure antibody concentrations and we therefore wrote anti-\ntetanus toxin IgG antibodies.\n4. Line 141: Bordetella pertussis should be italicized. Line 154: i.e. (as a Latin abbreviation) should also be\nitalicized.\nResponse: Changed\n5. Line 153: should read 0.1 IU/mL.\nResponse: Changed\n6. Line 170: should say “dashed lines” instead of “dotted lines.”\nResponse: Changed\n7. Line 195: sentence structure is awkward and should be revised for clarity.\nResponse: The sentence in Line 225-229 is changed to: The 40-59 years age group indicated a lower proportion of\nrecently infected individuals in the longitudinal serosurvey in October 2024 compared to the cross-sectional study\n(p=0.0028) (Supplementary Figure 4).\n8. Figure 3B: the double line of numbers under the x-axis is visually unappealing. The reviewer recommends\nkeeping only the even numbers (upper line), as it is clear that odd ages are included.\nResponse: Changed\n9. Lines 238–239: the measurement units are missing after “1.0” and “2.1.”\nResponse: Changed\n10. Line 298: the comma after “outbreaks” should be removed.\nResponse: Changed\n11. Line 314: should read CD4+ T.\nResponse: Changed\n12. Line 356: Pertactin (or pertactin, as the autors prefer) P.69\nResponse: Changed\n13. Line 360: should read “or anti-human IgA.”\nResponse: Changed\n14. Line 363: should read geometric mean.\nResponse: Changed\n15. Line 364: should read mean fluorescence intensity (MFI).\nResponse: Changed\n16. Line 366: delete the comma after “infection”.\nResponse: Changed\n17. Line 368: sentence should be in past tense.\nResponse: Changed\nLiterature\n1. Versteegen P, Berbers GAM, Smits G, Sanders EAM, van der Klis FRM, de Melker HE, et al.\nMore than 10 years after introduction of an acellular pertussis vaccine in infancy: a cross-sectional\nserosurvey of pertussis in the Netherlands. Lancet Reg Health Eur. 2021;10:100196.\n2. He Q, Viljanen MK, Olander RM, Bogaerts H, De Grave D, Ruuskanen O, et al. Antibodies to\nfilamentous hemagglutinin of Bordetella pertussis and protection against whooping cough in\nschoolchildren. J Infect Dis. 1994;170(3):705–8.\n3. A.M. van Roon SJL, H.E. de Melker. The National Immunisation Programme in the\nNetherlands. Surveillance and developments in 2023-2024. 2024.\n4. Vos Eric RA, van Hagen Cheyenne CE, Wong D, Smits G, Kuijer M, Wijmenga-Monsuur\nAlienke J, et al. SARS-CoV-2 Seroprevalence Trends in the Netherlands in the Variant of Concern Era:\nInput for Future Response. Influenza and Other Respiratory Viruses. 2024;18(6):e13312.\n5. EA van Lier JH, MR Holwerda, M Westra, H Giebers, NAT van der Maas, D van Winsen, IF\nZonnenberg-Hoff, JA van Vliet, HE de Melker. Vaccinatiegraad Rijksvaccinatieprogramma Nederland\nVerslagjaar 2025. 2025.\n6. Vos ERA, den Hartog G, Schepp RM, Kaaijk P, van Vliet J, Helm K, et al. Nationwide\nseroprevalence of SARS-CoV-2 and identification of risk factors in the general population of the\nNetherlands during the first epidemic wave. J Epidemiol Community Health. 2020;75(6):489–95.\n7. Moosa F, Tempia S, Kleynhans J, McMorrow M, Moyes J, du Plessis M, et al. Incidence and\nTransmission Dynamics of Bordetella pertussis Infection in Rural and Urban Communities, South\nAfrica, 2016‒2018. Emerg Infect Dis. 2023;29(2):294–303.\n8. Immink MM, Vos ERA, Janga-Jansen AVA, Baboe-Kalpoe S, Hulshof K, van Vliet J, et al.\nCirculation of Bordetella pertussis in the Caribbean Netherlands: a population-based\nseroepidemiological study. International Journal of Infectious Diseases. 2021;111:21–7.\n9. Visser LJ, van Veldhuizen J, Tromp V, Noomen R, Bonačić-Harpal A, Mariman R. Resurgence of\n<em>Bordetella pertussis</em> in the Netherlands in 2023-2024 reveals novel mutation in T3SS\ntranslocator gene <em>bopD</em>. medRxiv. 2025:2025.09.05.25335159.\n10. Kimura A, Mountzouros KT, Relman DA, Falkow S, Cowell JL. Bordetella pertussis filamentous\nhemagglutinin: evaluation as a protective antigen and colonization factor in a mouse respiratory\ninfection model. Infect Immun. 1990;58(1):7–16.\n11. Yougbare I, McTague A, He L, Choy CH, Su J, Gajewska B, et al. Anti-FIM and Anti-FHA\nAntibodies Inhibit Bordetella pertussis Growth and Reduce Epithelial Cell Inflammation Through\nBacterial Aggregation. Front Immunol. 2020;11:605273.\n12. Zeddeman A, van Schuppen E, Kok KE, van Gent M, Heuvelman KJ, Bart MJ, et al. Effect of\nFHA and Prn on Bordetella pertussis colonization of mice is dependent on vaccine type and\nanatomical site. PLoS One. 2020;15(8):e0237394.\n13. Warfel JM, Zimmerman LI, Merkel TJ. Acellular pertussis vaccines protect against disease but\nfail to prevent infection and transmission in a nonhuman primate model. Proc Natl Acad Sci U S A.\n2014;111(2):787–92.\nThank you for your positive response to our manuscript and for considering it for publication\nin Nature Communications. We appreciate the feedback provided by the reviewers and the\neditorial team and have addressed all remaining points. Below we response to each\ncomment of the reviewers. The changes made to the manuscript are shown using track\nchanges. The line numbers in our response refer to the track-changed version of the revised\nmanuscript.\nREVIEWERS' COMMENTS\nReviewer #1 (Remarks to the Author):\nIn this revised version, all comments have been taken into account. The manuscript has\nbeen significantly improved.\nReviewer #3 (Remarks to the Author):\nThe authors addressed my comments.\nMy major comment now is that authors need to discuss their findings in the context of known\nliterature. Please see more details below.\nComments:\nLines 113-114: \"Diagnostic confirmation of recent pertussis infection in paired samples\nnormally uses a >3-fold increase in anti-Ptx IgG concentration with a final concentration >20\nIU/mL \"- The authors should support this by data OR statements (please see the PMID:\n21069406 reference that comments on serological diagnosis of pertussis).\nResponse: We have added the suggested, and an additional reference and amended our\nstatement accordingly:\nLine 295-299: Diagnostic confirmation of recent pertussis infection in paired samples, with the\nsecond sample at two to four weeks interval, normally uses a >3-fold increase in anti-Ptx IgG\nconcentration with a final concentration >20 IU/mL (44-46). Here we used a ≥2-fold increase criterion,\ndue to a longer time interval between two measurements. We expected more waning of antibodies\nwithin that period, consequently leading to a lower fold-change between consecutive timepoints\nLines 122-124:\n\"vaccination. Because the Tdap booster vaccination contains reduced concentrations of the\npertussis components, and vaccination induces a weaker antibody response compared to\nnatural infection, a lower anti-Ptx IgG concentration cutoff 124 was used not to miss\nvaccination events. \" This must be supported by a reference.\nResponse:\nThank you for your comment. We based our rationale to use a lower cut-off for vaccination\non a previous report. Berbers et al. (1) compared the anti-Ptx IgG response following\nacellular pertussis vaccination and natural infection. They found that post-vaccination\nantibody concentrations were lower (peak geometric mean concentration after three different\naP vaccines: 78-90 IU/mL (lower bound of 90% interval (Q ): 9-27 IU/mL) vs 259 IU/mL\n0.05\nafter natural infection (Q : 110 IU/mL)). IU/mL values were calculated from EU/mL using a\n0.05\nfactor of 0.8, as previously described (2).\nPost-vaccination antibody concentration also showed a faster decay compared to natural\ninfection (geometric mean of 6-7 months compared to 17 months). Given our 6-month\nsampling interval, there is a possible longer time period between vaccination and serum\nsample. We therefore used a cut-off of 5 IU/mL to ensure the detection of a vaccination\nevent. We have added this reference to the sentence:\nBecause the Tdap booster vaccination contains reduced concentrations of the pertussis components, and\nvaccination induces a weaker antibody response compared to natural infection, a lower anti-Ptx IgG cutoff was\nused not to miss vaccination events (47). (Lines 306-309)\nThe discussion is good but a main concern is that it does not refer to , nor compare to,\nrecent papers that showed dynamics of pertussis antibodies during the COVID-19. Some of\nthese studies showed also higher than expected prevalence of pertussis infection. I highly\nrecommend that authors discuss their findings in the context of what has been reported (e.g.\nref: PMID: 36283895; PMID: 35746480; PMID: 35599039; PMID: 38797627).\nResponse: Thank you for your comment and suggestion of relevant papers. We agree that\nwe did not previously include recent papers on the dynamics of pertussis antibodies during\nthe COVID pandemic. We added statements and the recommended papers to our\ndiscussion in the following places:\nPMID 36283895 (He 2022) to:\nLines 150-151: This vaccine protects against infection for about 3-5 years after which individuals gradually\nbecome more susceptible (10-12).\nPMID 35599039 (Reicherz 2022) and 35746480 (Chen 2022) to:\nLines 151-156: We observed waning of anti-Ptx IgG antibodies in all age groups, as previously reported for\nadults (13,14). However the fastest waning was observed in children aged 2-5 years. The lowest anti-Ptx IgG\nconcentrations in those aged 6-12 years after the period of reduced circulation, which supports the finding of\nwaning immunity after the booster dose.\nPMID 38797627 (Reicherz 2024) to:\nLines 191-193: In contrast, a study from Canada found an increase in anti-Ptx IgG antibody concentrations\namong adults aged 25-51 between 2022 and 2023, suggesting recirculation of pertussis in this age group after\nthe lifting of COVID-19 NPIs (25).\nReviewer #4 (Remarks to the Author):\nRevision Report\nThe reviewer acknowledges the importance of this manuscript in advancing our\nunderstanding of population immunity against Bordetella pertussis, which is pivotal for\nimproving current immunization strategies and public health interventions to combat this\npathogen.\nThe authors have addressed both major and minor concerns raised by the reviewer, who\nrecognizes the complexity and the significance of the topic presented. As a means of\nconstructive communication, the reviewer would like to provide a few additional comments\non the authors’ responses, with the goal of helping them further strengthen their manuscript:\n1. Comment on vaccination coverage\nThe authors added the following clarification in the introduction:\n“Of special concern is the decrease in vaccination coverage in the Netherlands that has\nbeen observed over the past years, with 92.2% of the infants born in 2019 who have been\nfully vaccinated by the age of two (8). Since 2019, the maternal Diphtheria-Tetanus-acellular\nPertussis (Tdap) vaccination is implemented in the Dutch NIP to increase the level of\nmaternally-derived immunological protection in the first months post birth (2). However, this\nis not implemented globally and pertussis remains one of the least controlled vaccine\npreventable diseases.”\nThe recommendation was well addressed. As an additional suggestion, the reviewer\nrecommends verifying whether more recent data are available regarding the uptake of the\nthree-dose pertussis vaccine (administered as part of the hexavalent pediatric vaccine) in\nthe Netherlands. The phrase “with 92.2% of the infants born in 2019 who have been fully\nvaccinated by the age of two” lacks context—compared to what? Previous years? The last\ndecade?\nAccording to WHO data, the coverage for infants under one year of age in 2019 was\napproximately 94%, and this percentage has been steadily declining over the past decade,\ndropping from nearly 97% (2010) to about 91% (2024). Therefore, the reviewer suggests\ncorroborating the percentage reported by the RIVM in its annual publication with WHO data.\nResponse: The reviewer is correct that a comparison of vaccination in different years is\nmissing. We changed this sentence and compared the vaccination coverage in 2022 to the\nvaccination coverage from 2019 and 2010. We also added the WHO data as reference to\nthis.\nLine 51-53: The WHO and RIVM reported that the percentage of fully vaccinated infants by the age\nof two declined from 96-97% in the 2010 birth cohort to 92-94% in the 2019 birth cohort and 89-93%\nin the 2022 birth cohort (8,9).\n2. Comment on anti-FHA antibodies\nThe authors stated:\n“We agree that measuring antibody concentrations does not provide a complete picture of\nimmunity against pertussis, though earlier studies have already pointed out that antibodies\nagainst FHA correlate with protection against pertussis (2). Anti-FHA IgG concentrations\nplay an important role in prevention of colonization of pertussis (10–12).”\nThe reviewer agrees that some clinical and nonclinical studies have suggested a role for\nanti-FHA antibodies in protection against pertussis. However, other reports have found no\nsuch correlation (e.g., Cherry et al., 1998 (10.1016/S0264-410X(98)00226-6); Storsaeter et\nal., 1998; Lesne et al., 2020). FhaB has been associated with colonization of the upper\nrespiratory tract (Holubova et al., 2022) but appears to play no role in the lower respiratory\ntract (Zedderman et al., 2020). Espinosa-Vinals et al. (2025) also demonstrated that\nintranasal administration of an acellular pertussis vaccine containing PT and FHA provided\nlimited protection against lung colonization and no effect on nasal mucosal colonization or\ninfection (using full organ homogenates, as washes underestimate the bacterial load),\ncompared to formulations including Prn components.\nIncreasing evidence suggests that correlates of protection against pertussis might be more\nclosely linked to cellular immunity. Nevertheless, caution is advised, as the outcome being\nmeasured—protection from severe symptoms (e.g., leukocytosis, whooping cough) versus\nprevention of infection and/or colonization—matters greatly. To this reviewer, anti-FHA\nantibodies seem more like a surrogate marker, possibly predictive of protection but not\nnecessarily causally linked to immunity against the pathogen. The reviewer recommends\nexercising caution when making strong assertions on this point but truly respect the opinion\nof the authors in this regard.\nResponse:\nWe thank the reviewer for the comment on the complex role of anti-FHA in pertussis\nimmunity. We agree that several reports did not find a correlation between anti-FHA\nantibodies and protection, and we acknowledge the recommendation to be careful in making\nstrong assertions. In our discussion we aimed to reflect this by stating:\nLine 165-168: The lower anti-FHA IgG concentration in infected individuals prior to infection in the\ncurrent study suggests a potential protective role of anti-FHA antibodies in pertussis infection and\nprevention of colonization, however the precise role of anti-FHA antibodies is still topic of debate (19-\n20)\n3. Minor Corrections\nTable 1: While understandable, the reviewer recommends explicitly defining the term “3 Bp”\nin the table footnote (e.g., “triple antigen pertussis component” or another more accurate\ndescription of the author preference).\nResponse: The three Bp components are added to Table 2.\nLine 525: aP priming with three Bp components (Ptx, FHA and Prn)\nFigure 2: The labels should read “end.”\nResponse: Changed\nLiterature\n1. Berbers GAM, van de Wetering MSE, van Gageldonk PGM, Schellekens JFP,\nVersteegh FGA, Teunis PFM. A novel method for evaluating natural and vaccine induced\nserological responses to Bordetella pertussis antigens. Vaccine. 2013;31(36):3732–8.\n2. van der Lee S, Stoof SP, van Ravenhorst MB, van Gageldonk PGM, van der Maas\nNAT, Sanders EAM, et al. Enhanced Bordetella pertussis acquisition rate in adolescents\nduring the 2012 epidemic in the Netherlands and evidence for prolonged antibody\npersistence after infection. Eurosurveillance. 2017;22(47):17–00011.\nRevision Report\nThe reviewer acknowledges the importance of this manuscript in advancing our understanding\nof population immunity against Bordetella pertussis, which is pivotal for improving current\nimmunization strategies and public health interventions to combat this pathogen.\nThe authors have addressed both major and minor concerns raised by the reviewer, who\nrecognizes the complexity and the significance of the topic presented. As a means of\nconstructive communication, the reviewer would like to provide a few additional comments on\nthe authors’ responses, with the goal of helping them further strengthen their manuscript:\n1. Comment on vaccination coverage\nThe authors added the following clarification in the introduction:\n“Of special concern is the decrease in vaccination coverage in the Netherlands that has\nbeen observed over the past years, with 92.2% of the infants born in 2019 who have been\nfully vaccinated by the age of two (8). Since 2019, the maternal Diphtheria-Tetanus-acellular\nPertussis (Tdap) vaccination is implemented in the Dutch NIP to increase the level of\nmaternally-derived immunological protection in the first months post birth (2). However, this\nis not implemented globally and pertussis remains one of the least controlled vaccine\npreventable diseases.”\nThe recommendation was well addressed. As an additional suggestion, the reviewer\nrecommends verifying whether more recent data are available regarding the uptake of the\nthree-dose pertussis vaccine (administered as part of the hexavalent pediatric vaccine) in the\nNetherlands. The phrase “with 92.2% of the infants born in 2019 who have been fully\nvaccinated by the age of two” lacks context—compared to what? Previous years? The last\ndecade?\nAccording to WHO data, the coverage for infants under one year of age in 2019 was\napproximately 94%, and this percentage has been steadily declining over the past decade,\ndropping from nearly 97% (2010) to about 91% (2024). Therefore, the reviewer suggests\ncorroborating the percentage reported by the RIVM in its annual publication with WHO data.\n100\n) 99\n%\n( 98\ne 97\ns 96\no\nd 95 Administrative coverage\nd 94\nr Official coverage\n3 93\nP 92 WUENIC\nT 91\nD\n90\n2010 2012 2014 2016 2018 2020 2022 2024 2026\nYear\n2. Comment on anti-FHA antibodies\nThe authors stated:\n“We agree that measuring antibody concentrations does not provide a complete picture of\nimmunity against pertussis, though earlier studies have already pointed out that antibodies\nagainst FHA correlate with protection against pertussis (2). Anti-FHA IgG concentrations\nplay an important role in prevention of colonization of pertussis (10–12).”\nThe reviewer agrees that some clinical and nonclinical studies have suggested a role for anti-\nFHA antibodies in protection against pertussis. However, other reports have found no such\ncorrelation (e.g., Cherry et al., 1998 (10.1016/S0264-410X(98)00226-6); Storsaeter et al.,\n1998; Lesne et al., 2020). FhaB has been associated with colonization of the upper respiratory\ntract (Holubova et al., 2022) but appears to play no role in the lower respiratory tract\n(Zedderman et al., 2020). Espinosa-Vinals et al. (2025) also demonstrated that intranasal\nadministration of an acellular pertussis vaccine containing PT and FHA provided limited\nprotection against lung colonization and no effect on nasal mucosal colonization or infection\n(using full organ homogenates, as washes underestimate the bacterial load), compared to\nformulations including Prn components.\nIncreasing evidence suggests that correlates of protection against pertussis might be more\nclosely linked to cellular immunity. Nevertheless, caution is advised, as the outcome being\nmeasured—protection from severe symptoms (e.g., leukocytosis, whooping cough) versus\nprevention of infection and/or colonization—matters greatly. To this reviewer, anti-FHA\nantibodies seem more like a surrogate marker, possibly predictive of protection but not\nnecessarily causally linked to immunity against the pathogen. The reviewer recommends\nexercising caution when making strong assertions on this point but truly respect the opinion of\nthe authors in this regard.\n3. Minor Corrections\n Table 1: While understandable, the reviewer recommends explicitly defining the term\n“3 Bp” in the table footnote (e.g., “triple antigen pertussis component” or another more\naccurate description of the author preference).\n Figure 2: The labels should read “end.”"}]

Decision letter