Ten of 15 participants colonized after challenge with B1917 strain. Samples from 15 participants were collected between November 2017 and September 2018. Five participants were challenged intranasally with 1 × 104 colony forming units (CFU) B. pertussis strain B1917 and 10 participants were challenged intranasally with 1 × 105 CFU (Figure 1). As reported previously, participants challenged with 1 × 105 CFU more frequently reported mild symptoms of cough and rhinorrhea and nasal congestion than participants challenged with 1 × 104 CFU (12). None of the participants developed pertussis disease or required rescue medication. No serious adverse events were reported. To ensure that the different inoculum dose does not influence the immune cell kinetics, we first compared the kinetics of the 1 × 104 and 1 × 105 cohort. No statistically significant differences were observed between the cohorts, leading to the decision to merge these cohorts. The most prominent, but not statistically significant difference between the cohorts was in the number of plasma cells on day 11 (D11) and D14. This trend is indicated in the corresponding result section regarding B cell kinetics. One participant withdrew from the study after D14, and one participant withdrew after D28, both for reasons not related to the study. Samples collected until the moment of withdrawal were used for analysis.

Study and cohort description and clinical readout. Overview of study set up, intervention, and sampling time points used. Day 0 is the day on which participants were challenged intranasally. AB, antibiotics; BP, Bordetella pertussis.

To determine the colonization status after challenge, nasal washes were performed from D4 onwards as described previously (Figure 1) (12). Challenge with 1 × 104 CFU and 1 × 105 CFU resulted in B. pertussis–positive culture in 2 of 5 and 8 of 10 participants, respectively (Table 1). Of the 10 colonized participants, 4 showed low-density colonization (defined as <1000 CFU/mL at any time point during the study), and 6 showed high-density colonization (>1000 CFU/mL at any time point during the study) (Table 1). In further analyses, we divided the participants based on colonization and seroconversion status and searched for unique immune signatures associated with these readouts.

Clustering of participants based on the clinical readout (colonization status and seroconversion)

Ag-specific serum IgG levels (against PT, filamentous hemagglutinin [FHA], pertactin [Prn], and fimbriae 2 and 3 [Fim2/3]; expressed as IU/mL) were determined at baseline and on D28 (Figure 2). Comparison of baseline Ag-specific serum IgG levels of the 15 participants evaluated in this study versus all participants included in the overarching bacterial challenge study showed no differences (data not shown). Six participants showed signs of seroconversion (1 low-density- and 5 high-density-colonized participants), while participants protected against colonization showed no sign of seroconversion (Table 1). On top of increased anti-PT IgG serum levels, colonized participants also showed generally higher increases in other Ag-specific IgGs than participants protected against colonization (Figure 2). As only a greater than 2-fold increase in anti-PT serum IgG was used as cutoff for seroconversion, we also investigated whether any participant had a greater than 2-fold increase for another antigen. This was not observed. Lastly, the absolute increase in Ag-specific serum IgG was most prominent in high-density-colonized participants, especially for anti-PT and anti-FHA IgG (Figure 2).

Ag-specific serum IgG levels at baseline (D0) and 28 days after challenge (D28), as evaluated by multiplex immunoassay. Data are arranged according to degree of colonization. IU, international units; AU, arbitrary units; PT, pertussis toxin; FHA, filamentous hemagglutinin; Prn, pertactin, Fim2/3, fimbriae 2 and 3. n = 15 (5 colonization-protected, 4 low-density colonized, 6 high-density colonized).

Fluctuations in circulating innate immune cells after bacterial challenge. During natural encounter or challenge, the bacterium itself triggers innate immunity at the site of infection (mucosal surfaces); moreover, PT is known to induce systemic effects (13). Thus, a fast (and local) innate response may be important to control B. pertussis directly upon encounter. As recruitment of innate cells to and from tissues may be detected in the blood early after challenge, we evaluated the cellular changes of innate immune cell subsets after bacterial challenge.

Total numbers of circulating leukocytes and neutrophils (expressed as ratio vs. baseline) did not change after bacterial challenge (Figure 3, A and B). Circulating eosinophils decreased on D3 after challenge (Figure 3C). The most prominent changes were observed in circulating monocyte populations. Monocytes mature from classical monocytes (cMos) via intermediate (iMos) to nonclassical monocytes (ncMos) and can be further subdivided into different functional subsets or activation stages (14, 15). Fluctuations in total cMos were limited, with only a minor reduction in circulating cMos on D11 and D28 (data not shown). However, within cMo subsets, decreased numbers (ratio vs. baseline) were observed for CD62L+ cMos on D1, D4, D9, D11, D14, D28, and D56 after challenge, whereas increased numbers (ratio vs. baseline) were observed for CD62L– cMos on D1, D4, and D9 after challenge (Figure 3, D and E). CD62L, also known as L-selectin, is shed upon activation and CD62L– cMos are considered activated and possibly more mature (16). No consistent fluctuations were observed for iMos and ncMos (Figure 3, F and G). Within the dendritic cell (DC) compartment, no early consistent changes were observed, although myeloid DCs (mDCs) showed a decrease in cells on D28 after challenge (Figure 3, H and I).

Fluctuations in cell numbers in the innate immune cell compartment after bacterial challenge. Box-and-whisker plots (median, Q1, Q3, min-max) showing the kinetics of major innate immune cell subsets (A–I) expressed as ratio compared with baseline. n = 15. Dashed line indicates a ratio of 1.0 (baseline value). Baseline cell counts for the entire cohort, median (min-max) in cells/μL are indicated below each graph and merely added as an indication of frequency of the cell population. Longitudinal changes were evaluated using Wilcoxon’s matched-pairs signed-rank test and ratio versus baseline. Correction for multiple testing was performed using Bonferroni’s post hoc test. In case of significant differences in ratio compared with baseline, the median increase or decrease in cells/μL is indicated on top of the bar. (J–M) Increase or decrease in cell numbers expressed as ratio compared to baseline in colonized and colonization-protected participants. n = 15. Statistical test performed: Mann-Whitney U test on ratio of baseline. *P < 0.05; **P < 0.01; ***P < 0.001. D, days after challenge; cMo, classical monocyte; iMo, intermediate monocyte; ncMo, nonclassical monocyte; mDC, myeloid dendritic cell; pDC, plasmacytoid dendritic cell. Note: Although panel E shows that the ratio versus baseline was increased, the median cell count on D28 versus median cell count at baseline was decreased (–7 cells/μL).

To investigate whether the kinetics of innate immune cells are associated with colonization, we compared the numbers of circulating innate immune cells in participants who were colonized and participants who were protected against colonization. Similar to the collective kinetics, we assessed the longitudinal changes per cell population per colonization status. In order to avoid presenting an excess of data, only the time points where cell populations significantly differed from each other are shown in this paper. No consistent differences between colonized and colonization-protected participants were observed for leukocytes, neutrophils, eosinophils, iMos, cMos, and DCs. An increase in circulating ncMos (ratio vs. baseline), especially CD36– ncMos, was found on D1 after challenge in colonization-protected participants (Figure 3, J–L), while these were decreased in colonized participants. Finally, on D3 after challenge, colonization-protected participants had higher natural killer (NK) cell expansion (ratio vs. baseline) than colonized participants (Figure 3M).

Next, we divided the colonized participants based on colonization density (low- or high-density colonization). Here, we found that the decrease in (especially CD36–) ncMos was most prominent for low-density-colonized participants (Supplemental Figure 1, A–C; supplemental material available online with this article; https://doi.org/10.1172/JCI163121DS1). cMo maturation (represented by shedding of CD62L) did not differ between low- and high-density-colonized participants (data not shown). However, we did observe a decrease in a circulating mDC subset in low-density-colonized participants (CD1c+CD14dim mDCs; Supplemental Figure 1D). Lastly, NK cells in high-density-colonized participants did not expand, whereas the NK cell expansion in low-density-colonized participants to some extent resembled that of colonization-protected participants (Supplemental Figure 1E).

To summarize, we observed fluctuations in innate cell numbers in the early days after bacterial challenge. Decreased cell numbers may indicate migration of cells into the tissue, and the shedding of CD62L may indicate a shift toward a more activated phenotype in the cMo compartment. Cellular kinetics differed between colonization-protected and colonized participants, and between high- and low-density-colonized participants. The colonization density did not reflect the magnitude of cellular changes, as these were usually more prominent in low-density-colonized participants.

Limited fluctuations in T cell populations after challenge. Activation of T cells is required for cellular immunity and the activation of humoral immunity by providing T cell help to B cells in the germinal center reaction (17). As there are different T cell responses generated after vaccination with aP or wP and (natural) infection (18, 19), we set out to determine changes in circulating T cells after experimental exposure to B. pertussis.

Within circulating total T cells, CD4+ T cells, CD4+ Th, and regulatory T cells, no changes occurred until D28 and D56, when a significant decrease compared with baseline was observed on D28 for CD4+ T cells and regulatory T cells, and on D28 and D56 for total T cells (Figure 4, A–C). In follicular T helper (Tfh) cells, no changes were observed after challenge (Figure 4D). Additionally, no consistent changes were observed in CD4+ Th cell fluctuations (Figure 4, E–I) or Th cell maturation/activation (cell counts presented in Supplemental Table 1).

Fluctuations in cell numbers in the T cell compartment after bacterial challenge. Box-and-whisker plots (median, Q1, Q3, min-max) showing the kinetics of major innate immune cell subsets (A–I) expressed as ratio compared to baseline. n = 15. Dashed line indicates a ratio of 1.0 (baseline value). Baseline cell counts for the entire cohort, median (min-max) in cells/μL are indicated below each graph and merely added as an indication of frequency of the cell population. Longitudinal changes were evaluated using Wilcoxon’s matched-pairs signed-rank test and ratio versus baseline. Correction for multiple testing was performed using Bonferroni’s post hoc test. In case of significant differences in ratio compared with baseline, the median increase or decrease in cells/μL is indicated on top of the bar. (J–N) Increase or decrease in cell numbers expressed as ratio compared to baseline in colonized and colonization-protected participants. n = 13 (J–L) or n = 15 (M and N). Statistical test performed: Mann-Whitney U test on ratio of baseline. *P < 0.05; **P < 0.01; ***P < 0.001. D, days after challenge; Tfh, follicular T helper cells; Th, T helper cells; CXCR3–CCR4–CCR6+CCR10– = recently defined Th subset phenotype (20).

When grouping the participants based on colonization status, 3 different CD4+ Th subsets increased significantly in colonization-protected participants (ratio vs. baseline) compared with colonized participants on D1 after challenge (Figure 4, J–L). A small expansion in Th1/Th17 cells (CXCR3+CCR4–CCR6+CCR10–; recently described by Botafogo et al., ref. 20) was observed on D1 after challenge. Additionally, we found an expansion of Th22 cells and of the recently defined CXCR3–CCR4–CCR6+CCR10– Th subset on D1 in colonization-protected participants. The difference between colonized and colonization-protected participants was not fully explained by total CD4+ T cell kinetics (Figure 4M). An increase in total Tfh cells on D3 was primarily found in participants that were protected against colonization (Figure 4N). Due to technical and biological reasons (missing antibody in a surface stain master mix and/or CD45RA polymorphism; ref. 21), at this time point Tfh subsets could only be defined in 9 of 15 participants. Nevertheless, we observed a trend toward increased naive Tfhs, Th1-like, Th1/Th17-like, and Th17-like Tfhs on D3 after challenge in participants that were protected against colonization. This increase was significant for Th2-like Tfh cells (Supplemental Figure 2). When looking at the impact of colonization density, comparison between colonization-protected and low-density-colonized participants resulted in significant differences for Th cell subsets on D1 (Supplemental Figure 3, A–D). Moreover, Tfh expansion on D3 was higher in colonization-protected participants compared with both low- and high-density-colonized participants (Supplemental Figure 3E). No consistent changes were observed in maturation/activation of Th subsets.

Thus, although we observed consistent changes in the Th cell compartment on D1 after bacterial challenge, the most prominent change was the expansion of circulating Tfh cells on D3 after challenge. This expansion was only observed in participants protected against colonization and not polarized to any subset.

Increased plasma cell numbers on D3 and D11–D14 after challenge. T cell help, given by Tfh cells in the secondary lymphoid organs, is required to activate B cells after Ag encounter (22). Activation of B cells leads to the formation of memory B cells and plasma cells, and consequently to the production of (protective) antibodies. After (booster) vaccination with aP, skewing toward IgG1 plasma cells has been reported, whereas natural encounter is thought to induce IgA memory, as shown by the positive correlation between age and IgA responses against B. pertussis (23–26).

Upon challenge, fluctuations in the naive and memory B cell compartment were limited and not consistent between participants (Figure 5A, cell counts presented in Supplemental Table 1). However, plasma cells showed a trend toward increased cell numbers on D11–D14 after challenge (ratio vs. baseline); this expansion was observed in all isotypes (Figure 4, B–I, and Supplemental Figure 5A) and more prominent in participants inoculated with 1 × 105 CFU (Supplemental Figure 5B, trend). Of note, one participant (ID.12) showed strongly elevated (IgM) plasma cell counts at baseline. Therefore, data from this participant were not included in Figure 5, B–I. No consistent changes were observed in the distribution of maturation stages of total plasma cells, which were primarily defined by the loss of CD20 and gain of CD138 markers (Supplemental Figure 4D).

Kinetics in the plasma cell compartment upon bacterial challenge. (A) Box-and-whisker plot (median, Q1, Q3, min-max) showing the expansion of total memory B cells after challenge. n = 15. (B–I) Box-and-whisker plots (median, Q1, Q3, min-max) showing the expansion of total (B), IgM (C), IgG1–IgG4 (D–G), and IgA1 and IgA2 (H and I) plasma cells after challenge expressed as ratio compared to baseline (B–I: n = 14). Shown are medians and range. Dashed line indicates a ratio of 1.0 (baseline value). Baseline cell counts for the entire cohort, median (min-max) in cells/μL are indicated below each graph and merely added as an indication of frequency of the cell population. Longitudinal changes were evaluated using Wilcoxon’s matched-pairs signed-rank test on ratio versus baseline. Correction for multiple testing was performed using Bonferroni’s post hoc test. In cases of significant differences in ratio compared with baseline, the median increase in cells/μL is indicated on top of the bar. D, days after challenge. aParticipant ID.12 showed strongly elevated total and IgM plasma cell counts at baseline, which were approximately 5-fold (total plasma cells) and 25-fold (IgM plasma cells) higher compared with counts measured on D28 and D56. Therefore, this participant was excluded in panels B–I.

Next, we grouped the participants based on colonization status. Here, we observed an expansion of plasma cells on D3, which was most prominent in colonization-protected participants, but found in 7 participants in total (Figure 6A, trend). Among all plasma cell subsets, expansion on D3 seemed slightly more prominent for IgG1 (trend), IgG4 (trend), and IgA1 (trend). Plasma cell expansion on D11–D14 seemed unrelated to colonization status (Figure 6B). Next, we investigated the maturation patterns of IgG1 and IgA1 plasma cells in colonized and colonization-protected participants (Figure 6, C and D). We observed that in colonization-protected participants the expansion of IgG1 on D3 after challenge mostly consisted of the least and most mature IgG1 plasma cells, whereas for IgA1 this expansion consisted of the least and intermediate mature IgA1 plasma cells. For IgG4 plasma cells, cell counts were generally too low to reliably monitor all 3 plasma cell maturation stages. When assessing the distribution of total plasma cells over different maturation stages, we found a relative increase in more immature plasma cells on D3 and D4 after challenge in colonization-protected participants, but not in colonized participants (Figure 6C).

Different kinetics in the plasma cell compartment of colonized and colonization-protected participants. Note: Due to different normalization of plasma cells in participant ID.12, this participant was not included in the analysis of total plasma cells (panels A, B, and E; n = 14). (A) Plasma cell expansion on D3 after challenge, expressed as ratio compared to baseline. (B) Plasma cell expansion on D11–D14 after challenge expressed as ratio compared to baseline (of D11 and D14, the day of maximum expansion was used for each participant). n = 14. (C and D) Longitudinal changes in IgG1 (C) and IgA1 (D) plasma cell (PC) maturation stages expressed as ratio and min-max compared with baseline. n = 15. Dashed line indicates a ratio of 1.0 (baseline value). n = 15. Statistical test performed for longitudinal analysis: Wilcoxon’s matched-pairs signed-rank test on ratio of baseline. Correction for multiple testing was performed using Bonferroni’s post hoc test. Statistical test for comparison between groups per time point: Mann-Whitney U test on ratio of baseline. (E) Distribution of total plasma cells over 6 different maturation stages (S1–S6), expressed as percentage of total plasma cell population. Average plasma cell counts are indicated on the right side of each plot.

Then, we divided participants based on colonization density. Aside from colonization-protected participants, the plasma cell expansion on D3 was observed in 1 low- and 1 high-density-colonized participant (Supplemental Figure 5A). Expansion of plasma cells on D11–D14 after challenge was not specifically related to colonization density (Supplemental Figure 5B). Upon inspection of IgG1 and IgA1 plasma cells, we found that on D3 after challenge, colonization-protected participants had significantly higher numbers of the least mature IgA1 plasma cells, as compared with low-density-colonized participants. Moreover, both high-density-colonized and colonization-protected participants showed an increase in intermediate mature IgG1 and IgA1 plasma cells on D11–D14 after challenge (Supplemental Figure 5, C and D; trend). When assessing the distribution of total plasma cells over different maturation stages, the differences between low-density-colonized and colonization-protected participants were most prominent at early time points (D0, D3, and D4) (Supplemental Figure 5E).

As the expansion of plasma cells on D3 after challenge seemed more prominent in colonization-protected participants, we correlated the plasma cell expansion on D3 with the maximum CFU load and the CFU load on each day after challenge until the first day of azithromycin treatment. No correlation was found between the plasma cell expansion and the maximum CFU load. Instead, we found a negative correlation between plasma cell expansion on D3 and CFU load on D7 (Spearman’s r = –0.5773, P = 0.0269) and CFU load on D9 (Spearman’s r = –0.6215, P = 0.0157). Plasma cell expansion on D3 did not correlate with CFU load on D11 or D14. Moreover, as T cell help is required for B cell activation, we correlated the plasma cell expansion on D3 with Th cell expansion on D1 and Tfh expansion on D3, but no correlation was observed. Lastly, we correlated the plasma cell expansion on D3 with the anti-PT IgG levels at baseline. Although during initial screening anti-PT IgG levels greater than 20 IU/mL were used as an exclusion criterion, anti-PT IgG levels determined at actual baseline (second screening) in the participants ranged from 0.2–22.2 IU/mL, possibly suggesting a more recent B. pertussis encounter in some individuals. Indeed, we found a positive correlation (Spearman’s r = 0.6452, P = 0.01111) between these 2 values, indicating that cellular and humoral responses parallel each other.

Thus, within the B cell compartment, we observed most changes within the IgG and IgA plasma cell subsets, yet all were subtle and should therefore be confirmed in a larger study cohort. An early expansion of plasma cells on D3 after challenge was primarily observed in participants protected against colonization, whereas plasma cells on D11–D14 after challenge expanded irrespective of colonization status. A negative correlation was observed between CFU load and plasma cell expansion on D7 and D9 after challenge. Lastly, maturation of the IgG1 and IgA1 plasma cells was observed to some extent, especially toward an intermediate mature phenotype on D11–D14 after challenge.

Plasma cell expansion is a predictor of seroconversion. Ag-specific serum IgG levels are routinely used as a readout for vaccine efficacy and/or protective immunity; for example, anti-capsular IgG serum levels correlate with protection against Streptococcus pneumoniae (27). Therefore, we investigated cellular changes associated with seroconversion. Here, we found that all participants who seroconverted (1 low-density- and 5 high-density-colonized participants) had a more prominent expansion of total plasma cells on D14 after challenge compared with nonseroconverting participants (P < 0.05) (Figure 7A). This expansion was clearest in IgM, IgG1, and IgA1 plasma cells (P < 0.05). Yet, only a trend toward an increased number of intermediate (D14) and most mature (D11 and D14) IgG1 plasma cells was observed in participants who seroconverted compared with nonseroconverting participants (Figure 7B). For IgA1 plasma cells, a trend toward higher intermediate mature plasma cell counts was found in seroconverting participants on D11 and D14 (Figure 7C). When observing the distribution in maturation stages in the total plasma cell compartment, we observed a slight increase in more mature plasma cells in nonseroconverting participants on D3–D4 after challenge (Figure 7D).

Different plasma cell kinetics in participants that did or did not seroconvert. (A) Expansion of plasma cell numbers on D14 after challenge. Expansion is expressed as ratio compared to baseline. n = 15. (B and C) Longitudinal changes in IgG1 (B) and IgA1 (C) plasma cell maturation stages expressed as ratio and min-max compared with baseline. n = 15. Dashed line indicates a ratio of 1.0 (baseline value). n = 15. Statistical test performed for longitudinal analysis: Wilcoxon’s matched-pairs signed-rank test on ratio of baseline. Correction for multiple testing was performed using Bonferroni’s post hoc test. Statistical test for comparison between groups per time point: Mann-Whitney U test on ratio of baseline. *P < 0.05 (longitudinal change). (D) Distribution of total plasma cells over 6 different maturation stages (S1–S6), expressed as percentage of total plasma cell population. Average plasma cell counts are indicated on the right side of each plot.

Evaluation of other immune cell subsets did not reveal any additional cellular changes specific for seroconversion (cell counts presented in Supplemental Table 1). Lastly, we investigated the correlation between maximum colonization density in CFU during the study and the absolute increase in serum IgG (D28 – D0) directed against PT, FHA, Prn, and Fim2/3. We found a positive correlation for anti-PT IgG (Spearman’s r = 0.6486, P = 0.0109) and anti-FHA (Spearman’s r = 0.5684, P = 0.0296), but not for anti-Prn or anti-Fim2/3 IgG.

Baseline B. pertussis–specific serum IgG levels correlate negatively with maximum CFU count. It is known that an individual’s baseline immune status can impact the individual’s immune response. Therefore, we correlated the number of plasma cells at baseline and the B. pertussis–specific IgG serum levels with the maximum CFU load. Although anti-PT IgG serum levels greater than 20 IU/mL correlated with expansion of plasma cells on D3, they did not correlate with maximum CFU count (Spearman’s r = –0.2496, P = 0.3655). However, we found a negative correlation between B. pertussis–specific IgG levels and maximum CFU counts for anti-FHA (Spearman’s r = –0.5292, P = 0.0447), anti-Prn (Spearman’s r = –0.6170, P = 0.0166), and anti-Fim2/3 (Spearman’s r = –0.5210, P = 0.0487), possibly indicating preexisting immunity in several participants. No correlations were found between baseline plasma cell numbers and the maximum CFU load.

Most informative time points and cell populations associated with protection against colonization. This study identified the immune cell populations and sampling time points that are the most informative for follow-up studies. Especially in the early days after bacterial challenge (D1, D3, and D4), changes in the innate immune compartment were detected. Changes in the adaptive immune response can be monitored at various time points during the 2 weeks after challenge, with D1, D3, D11, and D14 being most informative. The most informative time points and cell populations to follow up on after bacterial challenge are summarized in Figure 8A (general kinetics after challenge) and Figure 8B (kinetics specific for colonization-protected participants).

Overview of the most informative time points and cell populations after bacterial challenge. (A) General cellular kinetics after challenge irrespective of clinical readout. (B) Cellular kinetics unique to participants protected against colonization, possibly indicating preexisting immunity in these participants.