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Published in final edited form as: Cell Immunol. 2016 May 14;304-305:35–43. doi: 10.1016/j.cellimm.2016.05.002

Th1 versus Th2 T cell polarization by whole-cell and acellular childhood pertussis vaccines persists upon re-immunization in adolescence and adulthood

Tara Bancroft a, Myles BC Dillon a, Ricardo da Silva Antunes a, Sinu Paul a, Bjoern Peters a, Shane Crotty a, Cecilia S Lindestam Arlehamn a, Alessandro Sette a,*
PMCID: PMC4899275  NIHMSID: NIHMS788886  PMID: 27212461

Abstract

The recent increase in cases of whooping cough among teenagers in the US suggests that the acellular Bordetella pertussis vaccine (aP) that became standard in the mid 1990s might be relatively less effective than the whole-bacteria formulation (wP) previously used since the 1950s. To understand this effect, we compared antibody and T cell responses to a booster immunization in subjects who received either the wP or aP vaccine as their initial priming dose in childhood. Antibody responses in wP- and aP-primed donors were similar. Magnitude of T cell responses was higher in aP-primed individuals. Epitope mapping revealed the T cell immunodominance patterns were similar for both vaccines. Further comparison of the ratios of IFNγ and IL-5 revealed that IFNγ strongly dominates the T cell response in wP-primed donors, while IL-5 is dominant in aP primed individuals. Surprisingly, this differential pattern is maintained after booster vaccination, at times from eighteen years to several decades after the original aP/wP priming. These findings suggest that childhood aP versus wP vaccination induces functionally different T cell responses to pertussis that become fixed and are unchanged even upon boosting.

Keywords: Epitope, pertussis, re-immunization, T cell polarization, vaccine

1. Introduction

Prior to the introduction of effective vaccines, rates of pertussis infection were high in the general US population (more than 150,000 cases per year from 1922–1950 [1]. Bordetella pertussis, the causative agent of whooping cough, infects the lungs and upper airways of human hosts. The infection leads to a persistent and paroxysmal cough sometimes associated with posttussive vomiting and apnea, and is associated with a mortality rate of 1–2%, most frequently among infants younger than 3 months of age [2]. The introduction of the inactivated whole-cell B.pertussis vaccine (wP, containing aluminum salts as adjuvant) in the 1950s led to a dramatic decrease in the number of cases and associated deaths. However, vaccine-related side effects provided a motivation to develop a safer vaccine construct [3]. The subsequently developed acellular vaccine (aP) consists of a mixture of purified antigens from the bacterium, including filamentous hemagluttinin (FHA), pertactin (PRN), pertussis toxin (PT), and fimbrial proteins 2 and 3 (Fim2/3), also formulated with aluminum salts. As the safety profile of aP was much improved, it quickly became the primary vaccine used in infants following approval in the US in the mid to late 1990s and is currently routinely used as booster vaccine for both adults and adolescents [4–6].

Unexpectedly, 28,660 cases of whooping cough were reported to CDC in 2014, and its incidence is expected to continue to rise by the end of 2015. Interestingly, the highest rates of increase were found in children and adolescents under the age of 20, but not in adults over the age of 20, corresponding to the first cohorts of infants that were aP-vaccinated in the mid 1990s that are now in their teenage years [7]. Accordingly, it is suspected that the switch to aP might be connected to this rise in infections [8–10].

Both aP and wP induce an increase in antibody titers against all aP components [11–14]. Adults originally primed with wP remain seropositive for multiple pertussis antigens up to 36 months following vaccination, with antibody titers at greater levels than at pre-vaccination [15]. However, protection against infection persists even after antibody titers have decreased, which suggests that low antibody titers may be protective for the entire inter-boost time, or that protection is due to a cell-mediated component of immunity to B. pertussis [16]. Animal studies suggest that memory CD4 responses to B. pertussis are required for long-lasting immunity to infection, and significant responses in these subsets can be detected after wP vaccination and after infection [17]. In humans, vaccination with aP induces a predominantly Th2 response, which may be associated with less effective protection from disease and infection [18, 19]; however, a reappraisal of the phenotype of T cells induced by vaccination is needed, since most of the seminal work in humans was performed before the appreciation of the substantial plasticity of Th1/Th2 subsets in humans [20, 21], and before the range of additional CD4 T cell subsets was recognized.

Several immunological mechanisms have been proposed to explain the decreasing efficacy of B. pertussis vaccination [8–10, 22], including the possibility that vaccination with aP induces a qualitatively different T cell response, at the level of which antigens and epitopes are recognized, and/or the phenotypes of T cells recognizing them. Surprisingly, a comparison of immunological parameters observed following aP booster immunization in teenagers originally primed in childhood with aP versus older adults originally primed with wP has not been performed. Characterization of responses to vaccine antigens and epitopes in the two epidemiological cohorts associated with differential disease incidence could inform future mechanistic studies. Accordingly, in the present study we assessed T cell and antibody responses to B. pertussis antigens and defined epitope repertoires using PBMCs from volunteers originally primed with either the wP or aP vaccine.

2. Materials and methods

2.1. Study subjects

We recruited 95 healthy adults originally primed with either aP or wP from San Diego, USA (Supplementary Table I). All participants provided written informed consent for participation in this cross-sectional study and clinical medical history was collected and evaluated. The current immunization schedule for pertussis in California is at 2, 4 and 6 months and then two times between 2–6 years and a booster immunization at 12 years and then every 10 years. Individuals, who have been diagnosed with B. pertussis infection at any given time in their life, were excluded. The remaining participants were divided in six different groups: aP- or wP-primed in the first years of life (according to the vaccination history and year of birth), wP-primed/aP-boosted or aP-primed/aP-boosted within 0.5–3 months or >3 months of donation (according to the vaccination history). All wP-primed/non-boosted and aP-primed/non-boosted donors had not, to the best of our knowledge, received recent Tdap booster immunizations at least 4 years prior to the beginning of this study. The aP-primed/non-boosted donors were all above 18 years of age and thus would have received their latest pertussis booster immunization approximately 6 years ago at 12 years of age. This study was performed with approvals from the Institutional Review Board at La Jolla Institute for Allergy and Immunology.

2.2. Peptides

Peptides were synthesized as crude material on a small (1 mg) scale by A and A (San Diego). Peptides were 785 16-mers overlapping by eight residues and spanning the five antigens included in the aP vaccines (FHA, Fim2/3, PRN, and PT) from the Tohama I and 18323 strains.

2.3. PBMC isolation and culture

PBMC were isolated from collected blood or leukopheresis by density gradient centrifugation, according to the manufacturer’s instructions. Cells were cryopreserved in liquid nitrogen suspended in FBS containing 10% (vol/vol) DMSO. Culturing of PBMCs for in vitro expansion was performed by incubating in RPMI (Omega Scientific) supplemented with 5% human AB serum (Gemini Bioscience), GlutaMAX (Gibco), and penicillin/streptomycin (Omega Scientific) at 2 × 106 per mL in the presence of individual aP antigens; FHA, PRN and formaldehyde fixed PT (Reagent Proteins), and Fim2/3 (List Biological Labs) at 5 µg/mL. Every 3 days, 10 U/ml IL-2 in media were added to the cultures.

2.4. ELISPOT assays

After 14 days of culture with individual aP components, the response to peptides, peptide pools, or whole antigens was measured by IFNγ and IL-5 dual ELISPOT as previously described [23]. Consistent with previous studies [24], to be considered positive, a peptide pool response had to match all of three different criteria. These three criteria were to elicit at least 100 spot-forming cells (SFC) per 106 PBMC, p ≤ 0.05 by Student’s t-test or by a Poisson distribution test, and stimulation index ≥ 2. Criteria for peptide positivity were identical except with a threshold of 20 SFCs per 106 PBMC was utilized.

2.5. HLA typing, restriction, and binding predictions

Donors were HLA typed either at the La Jolla Institute or by an ASHI-accredited laboratory at Murdoch University (Western Australia). Typing at LJI, for only Class II, was by next generation sequencing [25]. Specifically, amplicons were generated from the appropriate class II locus for exons 2 through 4 by PCR amplification. From these amplicons, sequencing libraries were generated (Illumina Nextera XT) and sequenced with MiSeq Reagent Kit v3 as per manufacturer instructions (Illumina, San Diego, CA). Sequence reads were matched to HLA alleles and donor genotyping assigned. HLA typing in Australia for Class I (HLA A; B; C) and Class II (DQA1; DQB1, DRB1 3,4,5; DPB1) was performed using locus-specific PCR amplification on genomic DNA. Primers used for amplification employed patient-specific barcoded primers. Amplified products were quantitated and pooled by subject and up to 48 subjects were pooled. An unindexed (454 8-lane runs) or indexed (8 indexed MiSeq runs) library was then quantitated using Kappa universal QPCR library quantification kits. Sequencing was performed using either a Roche 454 FLX+ sequencer with titanium chemistry or an Illumina MiSeq using 2 × 300 paired-end chemistry. Reads were quality-filtered and passed through a proprietary allele calling algorithm and analysis pipeline using the latest IMGT HLA allele database as a reference. Potential HLA-epitope restrictions were inferred using the RATE program [26].

2.6. ELISA assays for antibody titers

Nunc Maxisorp microtiter plates (Thermo Fisher Scientific) were coated with 0.65 µg/mL solution of PT, or a 1 µg/mL solution of either FHA, PRN, or Fim2/3 (Reagent Proteins and List Biological Labs) in carbonate buffer and incubated overnight at 4 °C. Plates were blocked with PBS with 0.1% Tween 20 (PBST) including 1% bovine serum albumin (BSA) for 90 min at room temperature. Sera were diluted initially at 1:200 and then serial dilution at 1:2 in PBST with 1% BSA. Sera were incubated on coated plates for 90 min at room temperature. Plates were washed six times with PBST. Secondary antibody was goat anti-human IgG labeled with horseradish peroxidase (Millipore AP113P) at 1:5000 in PBST with 1% BSA. 100 µL of OPD (Sigma) in 50 nM citrate buffer (pH 5.0) was used as enzyme substrate. The color reaction was terminated with 1 M hydrochloric acid. Optical density (OD) was measured at 490 nm. Total IgG antibody titers in international units (IU) were analyzed compared to the WHO serum reference standard (NIBSC; 06/140) for PT, FHA, and PRN. Fim2/3 titers were measured relative to the Fim2/3 IgG titer of the WHO standard in relative units (RU).

3. Results

3.1. Donor cohorts of individuals originally primed with the wP or aP vaccines

Differences in responses induced by original priming with wP versus aP, might contribute to the current increased incidence, especially in teenagers, of whooping cough. A direct comparison of aP and wP vaccination is not feasible in the USA because wP is no longer licensed. However, epidemiological data suggests that aP-primed, aP-boosted individuals are more susceptible to disease than those who are wP-primed and aP-boosted. Comparison of these two donor cohorts is thus directly relevant to reveal differences in vaccine efficacy.

Accordingly, in the initial set of experiments, we sought to characterize responses in individuals originally vaccinated with either wP (n = 13, born before 1991) or aP (n = 20, born after 1997), and that were not, to the best of our knowledge, boosted in the last 4 years, in terms of antibodies and T cells reactive to the four aP antigens (FHA, Fim2/3, PRN, and PT). Confirmation of aP vaccination were based on medical records (Supplementary Table I).

3.2. Similar magnitude and pattern of immunodominance in antibody responses of aP- versus wP-primed donors

To compare the magnitudes of antibody responses to the FHA, Fim2/3, PRN, and PT antigens, as well as relative responses among the four antigens, between the wP- and aP-primed cohorts, serum from each donor was assayed by ELISA. The sum of antibody responses to all antigens is shown in Figure 1A. Sizeable antibody titers were detected in individuals immunized with either wP or aP; the titers did not differ significantly between the two cohorts.

Fig 1.

Fig 1

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Magnitude and immunodominance of antibody and T cell responses are similar between wP- and aP-primed cohorts. (A) Sum of titers across all aP antigens from each donor in wP/no boost and aP/no boost cohorts, as assayed by ELISA. (B) Immunodominance of antigens in both cohorts expressed as the percentage of the total response. (C) Overall responses against aP peptides as measured by dual color ELISPOT. Each data point represents the sum of responses across all aP antigens for a single wP/no boost or aP/no boost donor. (D) Immunodominance of each antigen is expressed as percentage of the total T cell response in wP and aP cohorts. Data is expressed as median ± interquartile range for each cohort. *: p < 0.05, ns, no significant difference by Mann-Whitney test.

In the wP-primed donors, responses were spread over all four antigens, with PT, PRN, FHA, and Fim2/3 each accounting for 20–33% of the total response (Figure 1B). A similar pattern was detected in the aP-primed donors (Figure 1B), with the four antigens each accounting for 16–27% of the total response. Thus, we did not observe major differences in either antibody titers or antigen dominance between the wP -and aP-primed cohorts.

3.3. T cell responses are higher in aP-primed compared to wP-primed donors

In preliminary experiments weak or undetectable responses were observed with PBMCs in response to ex vivo stimulation with either whole PT antigens or peptides. By contrast, vigorous responses could be detected in PBMCs from the aP- and wP-primed cohorts cultured for 14 days with whole antigen and in the presence of IL-2. The magnitude of T cell responses, obtained by summing all positive responses to individual peptides in a given donor, is shown in Figure 1C. A sizeable response was detected in individuals originally primed with wP (median 2900 SFC/106 PBMC per donor, interquartile range (IQR) 830 to 4400). The response magnitude was approximately 2-fold greater in the aP-primed cohort (median 6800 SFC/106 PBMC per donor, IQR 3700 to 15500; p=0.01). For both donor cohorts responses were spread over all four antigens, with FHA being most dominantly recognized (37–50% of the total response) (Figure 1D). PRN and PT accounted for similar proportions of T cell responses in both aP- and wP-primed donors (23% and 24%, and 32% and 20% respectively). Responses to Fim2/3 were minor (11% in wP-primed and 3% in aP-primed donors).

In comparison to the pattern of dominance observed for antibody titers, FHA is more dominant for T cell recognition, and conversely Fim2/3 is less recognized. This difference suggests that T cell and antibody responses for the aP vaccine antigens are independently regulated.

3.4. Antibody and T cell responses from aP- and wP-primed cohorts are both increased three months following aP booster vaccination

We next analyzed samples derived from donors that were originally primed with either aP or wP, and boosted with aP within the previous 3 months (Supplementary Table I). Donors that were boosted more than 3 months ago were excluded from this analysis.

We compared antibody and T cell responses to all aP antigens between aP-primed/aP-boosted and wP-primed/aP-boosted donors. In both donor cohorts, as expected, antibody responses increased after the aP boost (Figure 2A). Similar to the wP- and aP-primed donors, in the wP-primed/aP-boosted donors, responses were spread over all four antigens, with PT, PRN, FHA, and Fim2/3 each accounting for 16–34% of the total response (Figure 2B). A similar pattern was detected in the aP-primed/aP-boosted donors (Figure 2B), with the four antigens each accounting for 22–31% of the total response. Antibody responses to each individual antigen also increased in both donor cohorts after the aP boost (Figure 2C–F). Likewise, T cell responses in wP-primed individuals were significantly increased after boost. However, in aP-primed individuals, boosting only marginally increased pertussis-specific T cell frequencies (Figure 3A). Notably, pertussis-specific memory T cells were present at significantly higher frequencies at baseline in the aP group than the wP group (Figure 3A). For both donor cohorts responses were spread over all four antigens, similar to wP- and aP-primed donors, with PRN being most dominantly recognized (36–43% of the total response) (Figure 3B). FHA and PT accounted for similar proportions of T cell responses in both aP- and wP-primed donors (26% and 29%, and 28% and 26% respectively). Responses to Fim2/3 were minor (9% in wP-primed and 3% in aP-primed donors). T cell responses to individual antigens in aP-primed/aP-boosted donors were only significantly increased to PRN, while there was a significant increase in response to both PRN and PT in the wP-primed/aP-boosted donors (Figure 3C–F).

Fig 2.

Fig 2

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aP boosting increases B cell responses in wP- and aP-primed donors. (A) Sum of antibody titers for all aP antigens with or without aP boost (0.5–3 months post boost). wP (circles) and aP (squares). (B) Immunodominance of antigens in both wP/aP and aP/aP cohorts expressed as the percentage of the total response. (C–F) Sum of antibody titers for individual aP antigens with or without aP boost (0.5–3 months post boost). wP (circles) and aP (squares). (C) FHA, (D) Fim2/3, (E) PRN and (F) PT. Data are expressed as median ± the interquartile range for each cohort. *: p<0.05, **: p<0.01, ***:p<0.001, ****:p<0.0001, ns: no significant difference by Mann-Whitney test.

Fig 3.

Fig 3

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aP boosting increases T cell responses in wP-primed donors. (A) Sum of T cell responses for all aP antigens with or without aP boost (0.5–3 months post boost). wP (circles) and aP (squares). (B) Immunodominance of antigens in both wP/aP and aP/aP cohorts expressed as the percentage of the total response. (C–F) Sum of T cell responses for individual aP antigens with or without aP boost (0.5–3 months post boost). wP (circles) and aP (squares). (C) FHA, (D) Fim2/3, (E) PRN and (F) PT. Data are expressed as median ± the interquartile range for each cohort. *: p<0.05, ns: no significant difference by Mann-Whitney test.

3.5. Similar epitope repertoires recognized by aP-primed and wP-primed individuals

As the difference in protection between wP and aP priming could relate to the associated epitope repertoires, the reactivity of PBMCs to pools of T cell epitopes was measured and positive pools were deconvoluted. Overall, T cell responses were analyzed for a total of 49 aP-primed and 46 wP-primed individuals (both boosted; 0.5–44 months ago, and non-boosted). Positive responses were detected for a total of 601 peptides. As shown in Figure 4A, over 90% of donors recognized at least one epitope, and 50% of the donors responded to ≥10 epitopes, regardless of initial priming vaccination. By this analysis the breadth of responses associated with wP is marginally broader that those associated with the aP vaccination.

Fig 4.

Fig 4

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Similar epitope repertoires recognized by aP-primed and wP-primed individuals. (A) The proportion of each donor cohort who respond to the indicated number of epitopes, wP squares and aP circles. (B) Epitopes ranked on the basis of magnitude of response. wP; black line, aP; dashed line. Dotted lines indicate the top 50 and 100 epitopes. (C) Epitopes ranked on the basis of the response frequency for wP and aP combined. Black dotted line indicates the top 132 epitopes recognized by >5% of donors. (D) Overlap of top 132 epitopes recognized in 5 or more individuals in wP- and aP-primed cohorts.

The top 50 epitopes in each cohort accounted for 55–65% of the total response and the top 100 epitopes for 70–80% of the total response (Figure 4B). A total of 132 epitopes were recognized in at least 5 donors, corresponding to a frequency of recognition of 5.3% (Figure 4C).

The epitope repertoire recognized by the aP- and wP-primed donors was largely overlapping, with a total of 124/132 of the dominant epitopes recognized in both cohorts (Figure 4D). These 132 epitopes, together with their antigen of origin, total SFC/106 detected, and response frequency is listed in Supplementary Table II.

3.6. Inferred HLA restriction of dominant epitopes

To facilitate correlation of HLA types with T cell responses and the design of tetramer reagents to detect antigen-specific T cells, we used the RATE program [26] to calculate the relative frequency and significance of association between all the epitopes/regions and HLA alleles (or combinations thereof) expressed in responding donors.

This analysis allowed inferring potential restrictions for 97 of the main epitopes, of which 93 were promiscuous restrictions (the epitope was inferred to be restricted by multiple HLAs), thus confirming and extending previous results that suggested that dominant epitopes are often associated with promiscuous HLA restriction [27].

Supplementary Table III lists for each of these peptides the inferred restrictions, and details the number of donors that responded (R+) or did not respond (R−) to a given peptide, and the number of donors expressing (A+) or not expressing (A−) a given HLA(s). Accordingly, for example, the GADLIIANPNGISVNG epitope, 35 % (8/23) of the responders express the HLA molecules DRB1*14:04,DRB1*04:01/:04 and/or DRB4*01:07, while only 3/71 (4%) of the non-responders expressed the same HLAs (p= 0.0004).

3.7. Differential polarization of T cell responses as a function of the original priming persists for more than 18 years, despite similar boosting regimens

aP vaccination has been reported to induce greater responses of the Th2 subset than wP, and this Th2 bias is maintained in 4–6-year-olds boosted with aP [28]. However, this bias does not lead to a lack of protection; the aP vaccine is efficacious in preventing pertussis infections in infants and children up to 5 years old [29].

Here we sought to examine whether this bias is maintained into adolescence and adulthood. Accordingly, we examined the polarization of T cell responses, as measured by the ratio of IL-5/IFNγ responses. As shown in Figure 5A, among donors not receiving a recent booster immunization the dominant cytokine secreted upon stimulation with the epitope pools was IFNγ in wP donors, (median 5, IQR 2 to 31) while in aP donors, responses were biased towards IL-5 production (median 0.4, IQR 0.1 to 2.5). This bias was maintained even after recent boosting with aP; recently boosted wP/aP donors maintained IFNγ polarization (Figure 5A. Median 24, IQR 14 to 54), and recently boosted aP/aP donors maintained IL-5 responses (median 0.07, IQR 0.005 to 0.3). This results in an approximately 300-fold difference in the IFNγ/IL-5 pertussis-specific T cell ratio between wP/aP donors and aP/aP donors after booster immunizations.

Fig 5.

Fig 5

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Differential polarization of T cell responses. (A) The number of IL-5 and IFNγ SFCs were measured by dual color ELISPOT assays. Each data point represents the ratio of IFNγ/IL-5 SFCs from each donor; bars represent the median ± interquartile range. ****: p < 0.0001 by Mann-Whitney test. (B) Ratio of IFNγ to IL-5 secreting cells as a function of age. Each data point represents the log of the IFNγ/IL-5 SFC ratio from each donor. The best fit of each data set is represented by linear regression lines; R-squared and p values are shown.

Inherent in the design of our study is the fact that the different donor cohorts are of different median ages, which could affect the balance of Th1 and Th2 responses. As detailed in Supplementary Table I the median age of the wP-primed/aP-boosted cohort was 27 (24 to 65), and the median age of the aP-primed/aP-boosted cohort 19 (18–21). To investigate the effect of these age differences, we plotted response polarization as a function of age in each of the two cohorts (Figure 5B). Th1/Th2 polarization did not correlate with age in either cohort, suggesting that age does not likely account for the difference in IL-5/IFNγ ratios between wP- and aP-primed cohorts.

These data demonstrate that the significant difference in Th1/Th2 polarization of responses as a function of original pertussis vaccine priming is maintained for years, and is still prominent even following booster with the aP vaccine.

4. Discussion

Here we report an analysis of the T cell and antibody responses detected in late adolescent and adults, as a function of childhood priming with either acellular or whole-cell pertussis vaccine. Our analysis was performed 18 years to several decades after this original vaccination, and also in the context of recent boosting with aP. Both the wP and aP are effective vaccination strategies in childhood. However the increase in disease incidence in teenagers and young adulthood suggests that the immunity wanes overtime, suggesting that indeed aP vaccination is not as protective as wP but only or more at the level of the teenager population. Therefore it also highlights the importance of our research design that studies immune reactivity in teenagers and adults but not in children.

Previous reports have shown that the aP and wP vaccines induce differential T cell polarization, with the aP vaccine leading to a predominant Th2 response, while the Th1 component dominates the responses elicited by wP vaccination [18, 19, 28, 30–33]. All these studies were conducted either using samples from infants (<1 year old) [30, 31], young children (4–6 years old) [19, 28, 32] or pre-teenagers (9–12 years old) [33]. In that regard, our study was designed to encompass young adults and adults to better understand the persistence of long lasting immunizations. Unexpectedly, we see here that this Th1 bias is stubbornly maintained for decades - possibly lifelong - even after boosting with the Th2-biasing aP vaccine. It would thus appear that, once the immune response is polarized towards a Th1 phenotype, this polarization is faithfully conserved. Examining production of other cytokines is also of interest. In particular it will be of interest for future studies to measure other Th2 cytokines such as IL-4, or IL-10 because its possible involvement in Treg activity, or IL-17 as this cytokine has been linked to potential protective effects from whooping cough disease. Furthermore, in the present study we did not measure cytokine production by ELISA, but based on the study of Ryan et al., [28] expected to correlate with SFC counts.”

These findings are reminiscent of the “original antigenic sin” effect, originally reported in the case of influenza infections. Our findings are novel in that they suggest that, unlike influenza, in pertussis the “original sin” is acting not at the level of which epitopes are recognized, but rather at the level of the differentiation program imbued upon the responding T cells. This further suggests potential practical applications. It might be possible to devise “polarizing” adjuvants or regimens to correct the efficacy shortcomings of aP vaccines. More generally the data suggest that a polarizing immunization could result in a life-long bias. We cannot exclude that some donors may have had subclinical infection or exposure to crossreactive bacteria. However, both of these should occur equally in both aP and wP cohorts and despite this the polarizing immunization bias is maintained. Thus, this implies that it might be possible to immunize at-risk populations with allergens to induce a Th1-polarizing response and prevent the development of childhood and adolescent allergies.

The focus of our study was on analysis of T cell responses. However, it should be pointed out that determination of isotypes, affinity or opsonization mediated by Pertussis-specific antibodies might provide additional insights into the mechanisms involved in the differential efficacy provided by the two vaccines. We hypothesize that the aP vaccine would induce IgG4 due to the Th2 polarization.

Our analysis also addresses certain mechanisms that might explain differential vaccine efficacy. Specifically, based on our data it is unlikely that differences in the patterns of immunodominance at the antigen level or decreased magnitude of either T cell or antibody responses can explain the resurgence of infection. Likewise, differences in the type of epitope recognized or the breadth of epitope repertoire appear not be related to the longer efficacy of wP. This observation appears to support earlier findings that suggest that long-term multi-epitope specificity is lost with age [34].

In practical terms the present study also greatly expands our knowledge of human T cell epitopes derived from aP antigens. Here, we defined over 100 different dominantly recognized epitopes, the vast majority of which are novel. Our studies also re-identified 4 out of the 5 known pertactin epitopes, and 19 out of the 32 known PT epitopes, and the only FHA epitope thus far known [34–37]. Furthermore we report here the first identification of human T cell epitopes derived from the Fim2/3 proteins. Importantly HLA restrictions could be inferred for several of these epitopes. In the case of the DRB1:0101 restricted YYSNVTATRLLSSTNS from PTxB129–144 epitope, this inference was tested by tetramer staining experiments and found to be correct [26]. Thus we predict that this study will enable broad and accurate characterization of T cell responses to aP antigens.

A limitation of this study is that we have not characterized T cell responses elicited by wP vaccination and directed against other PT antigens not included in the aP vaccine. Indeed other pertussis-antigens, not used in this study, have been described in a recently published paper, including two envelope proteins, i.e., putative periplasmic protein (PPP) and putative peptidoglycan-associated lipoprotein (PAL), and two cytosolic proteins, i.e., 10kDa chaperonin groES protein (groES) and adenylosuccinate synthetase (ASS) [38]. It is possible that these antigens could contribute to the pertussis-specific immune response in wP-primed individuals. Additionally, responses detected in wP immunized individuals may be significantly stronger if whole bacteria were used as an antigen. However, this is not devoid of technical challenges since inactivation of the bacterial lysate may alter both T and B cell responses. Future studies utilizing a genome-wide screen approach will be required to identify which additional B. pertussis antigens are targeted by T cell responses, similar to what was done for M. tuberculosis [27].

Another important consideration is that in the present study we have characterized only Th1 and Th2 responses, and a more thorough characterization of responses, including memory markers, chemokine receptors and other cytokines will be of considerable interest, enabled by the current epitope identification studies. It is possible that the differential vaccine efficacy might be related to differential induction of Tregs. However, in pilot experiments IL-10 was not detected in our ELISPOT assays, suggesting that IL-10 producing regulatory T cells play a minor role, in the response to aP antigens. It is also possible that an environmental or developmental trigger encountered after childhood and in the teenage years, or age related changes in the immune system, might be related to differential vaccine efficacy. These possibilities will have to be addressed in future studies.

Overall these studies might shed light on the mechanism by which the aP and wP vaccines lead to different polarization and how it might impact in protective efficacy.

Supplementary Material

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2
3

Highlights.

  • Epitope repertoires recognized by aP- and wP-primed donors are similar.

  • Magnitude of T cell responses is higher in aP-primed donors.

  • IFNγ strongly dominates the T cell response in wP-primed donors, whereas IL-5 is dominant in aP-primed individuals.

  • Differential pattern of polarization is maintained even after booster vaccination.

Acknowledgments

The authors thank Laura Dullanty and John Pham for technical help, Alyssa Hill and Annie Lei for recruiting research subjects, John Sidney for technical advice, Ravi Kolla for help towards launching the project, and Jessica Moore for editing the manuscript.

T.B., M.B.C.D., B.P., S.C., R.d.S.A., C.S.L.A., and A.S. participated in the design and planning of the study. T.B., M.B.C.D., and C.S.L.A. performed and analyzed experiments. S.P. performed bioinformatical analyses. T.B., B.P., S.C., C.S.L.A., R.d.S.A., and A.S. wrote the article. All authors have read, edited, and approved the manuscript.

This work was supported by National Institutes of Health [contract HHSN272200900044C and U19 AI118626 to A.S.].

Footnotes

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Conflict of Interest

The authors have no conflict of interest.

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Associated Data

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Supplementary Materials

1
NIHMS788886-supplement-1.xlsx (43.2KB, xlsx)
2
NIHMS788886-supplement-2.xlsx (68KB, xlsx)
3
NIHMS788886-supplement-3.xlsx (19.2KB, xlsx)

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