- Methodology article
- Open Access
Serum reactome induced by Bordetella pertussis infection and Pertussis vaccines: qualitative differences in serum antibody recognition patterns revealed by peptide microarray analysis
- Davide Valentini†1, 2,
- Giovanni Ferrara†3, 4, 5,
- Reza Advani6,
- Hans O Hallander6 and
- Markus J Maeurer1, 2Email author
© Valentini et al.; licensee BioMed Central. 2015
Received: 12 June 2014
Accepted: 31 March 2015
Published: 1 July 2015
Pertussis (whooping cough) remains a public health problem despite extensive vaccination strategies. Better understanding of the host-pathogen interaction and the detailed B. pertussis (Bp) target recognition pattern will help in guided vaccine design. We characterized the specific epitope antigen recognition profiles of serum antibodies (‘the reactome’) induced by whooping cough and B. pertussis (Bp) vaccines from a case–control study conducted in 1996 in infants enrolled in a Bp vaccine trial in Sweden (Gustafsson, NEJM, 1996, 334, 349–355).
Sera from children with whooping cough, vaccinated with Diphtheria Tetanus Pertussis (DTP) whole-cell (wc), acellular 5 (DPTa5), or with the 2 component (a2) vaccines and from infants receiving only DT (n = 10 for each group) were tested with high-content peptide microarrays containing 17 Bp proteins displayed as linear (n = 3175) peptide stretches. Slides were incubated with serum and peptide-IgG complexes detected with Cy5-labeled goat anti-human IgG and analyzed using a GenePix 4000B microarray scanner, followed by statistical analysis, using PAM (Prediction Analysis for Microarrays) and the identification of uniquely recognized peptide epitopes.
367/3,085 (11.9%) peptides were recognized in 10/10 sera from children with whooping cough, 239 (7.7%) in DTPwc, 259 (8.4%) in DTPa5, 105 (3.4%) DTPa2, 179 (5.8%) in the DT groups. Recognition of strongly recognized peptides was similar between whooping cough and DPTwc, but statistically different between whooping cough vs. DTPa5 (p < 0.05), DTPa2 and DT (p < 0.001 vs. both) vaccines. 6/3,085 and 2/3,085 peptides were exclusively recognized in (10/10) sera from children with whooping cough and DTPa2 vaccination, respectively. DTPwc resembles more closely the whooping cough reactome as compared to acellular vaccines.
We could identify a unique recognition signature common for each vaccination group (10/10 children). Peptide microarray technology allows detection of subtle differences in epitope signature responses and may help to guide rational vaccine development by the objective description of a clinically relevant immune response that confers protection against infectious pathogens.
Pertussis (whooping cough) caused by B. pertussis (Bp), remains a major global public health problem [1,2]. Despite a vaccine coverage over 90% in newborns, pertussis remains endemic in the Western countries . In the first months of 2010, outbreaks have been described in Ireland , Israel  and USA . In California a new outbreak in 2014 was particularly severe, with 10.831 reported cases from January 1st to December 31st  (the worst toll since 1947).
The efficacy of current vaccination programs is likely hampered by adaptation of the pathogen, overcoming the effect of herd immunity . A comprehensive study covering Bp clinical isolates from 1935 to 2004 showed the appearance of a Bp strain that carries a mutation in the pertussis toxin promoter; the increased expression of this virulence factor directly correlated with the resurgence of pertussis in the last decades in the Netherlands . Another study from the same country, covering the period 1965 to 1992, showed the circulation of different serotypes of the pathogen in correlation with the use of whole cell or acellular pertussis vaccines in different time-frames . Substantial evidence has been accumulated in the last two years that immunity induced by acellular vaccines is much shorter lived than immunity induced by whole cell vaccines .
There is an unmet need i) to depict the immunological recognition matrix to understand the specific epitope recognition pattern induced by natural infection with Bp, ii) to identify differences in immune recognition induced by available Bp vaccines as compared to natural infection, and iii) to objectively define the qualitative differences in humoral target recognition induced by current vaccines . We assessed in the current study the immune recognition pattern in serum from infants with whooping cough and in 3 groups of infants randomized to different Bp vaccines from a trial conducted 1996 in Sweden  using a high-content peptide microarray. The immune recognition profile (or ‘reactome’) represents a detailed molecular recognition ‘fingerprint’ of serum IgG directed against linear epitopes.
Material and Methods
10 children who received a diphtheria (D) and tetanus (T), vaccine (DT, produced by Swedish National Bacteriological Laboratory, Stockholm, Sweden) as placebo, and developed whooping cough;;
10 children immunized with the diphtheria (D), tetanus (T), pertussis (P) whole cell (wc) (DTPwc) vaccine licensed in the United States (Connaught Laboratories, Swiftwater, PA, USA);
10 children immunized with the 5 component acellular candidate DTPa5 vaccine (Connaught Laboratories, Toronto, Canada);
10 ichildren immunized with the 2 component acellular candidate DTPa2 vaccine (SmithKline Beecham, Rixensart, Belgium);
10 children immunized with the Swedish-produced DT vaccine and did not develop whooping cough.
Sera were collected 30 days after the last dose, except for the group which whooping cough (group 1, convalescence sera).
The Stockholm regional ethics committee North (Dnr 911258) has approved the study. All subjects provided informed consent. Both parents of the children provided informed consent on their behalf. The informed consent was provided in a written format, signed and is on file at the Swedish National Institute of Public Health, Stockholm, Sweden.
Microarray slides and experiments
B. pertussis proteins spotted on the peptide microarray slides
Pertussis toxin subunit 1 precursor, (ptxA)
Pertussis toxin subunit 2 precursor, (ptxB)
Pertussis toxin subunit 3 precursor, (ptxC)
Pertussis toxin subunit 4 precursor, (ptxD)
Pertussis toxin subunit 5 precursor, (ptxE)
P.69 protein (pertactin/PRN)
DTPa5 & DTPwc
Filamentous hemagglutinin (FHA)
DTPA2,5 & DTPwc
Fim2 pilic subunit (Fim2)
Fim3 pilic subunit (Fim3) precursor
Tracheal colonization factor (TCF)
Bifunctional hemolysin-adenylate cyclase precursor (ATC/cyaA)
Outer membrane porin protein precursor (OMP)
Outer membrane porin protein
Outer membrane porin protein (OmpQ)
Outer membrane porin protein
GTP-binding elongation factor (BipA)
Bordetella resistance to killing (BrkA)
Vag8 protein (autotransporter) (Vag8)
Putative autotransporter (BapC)
Experiments were performed following a standardized protocol [14-16]: 300 μL serum diluted 1/100 in washing solution (filtered PBS, 3% fetal calf serum, FCS, Lot nr 45K3397, Sigma, Munich Germany and 0.5% Tween) were pipetted on the peptide microarray slide, covered with a cover slip (Gene-Frame, Abgene, UK) to evenly distribute the dilution over the slide and incubated at +4°C in a humid chamber for 16 hours; after the removal of the cover slip, the slides were washed 5 times on a shaker for 5 min each (twice with washing solution, twice with sterile water and one wash with filtered Milli Q water at the end).After washing, 300 μL Cy5-labeled goat anti-human IgG, affinity purified secondary antibody (Abcam, UK) diluted 1/500 in the washing solution were added (in the dark), and incubated in the dark 1 hour in a humid chamber at room temperature. Washing steps were repeated after the incubation with the secondary antibody. Prior to scanning, slides were dried with a slid spinner (Euro Tech, UK). Five additional slides were processed using only buffer in the first incubation step, to detect false positive spots due to non-specific binding of the secondary reagent. High-definition images from the slides were acquired with GenePix 4000B microarray scanner (Axon Instruments-Molecular Devices, Union City, US) using the wavelength 635 nm (red channel, for the specific IgG signal quantification) and 532 nm (green channel, positive controls for grid alignment and orientation). Data acquisition from the images was performed with the software Gene Pix 6 Pro (Axon Instruments-Molecular Devices, Union City, US).
Data analysis consisted of 4 steps as described .
All images and aligned files were visually inspected to check for artifacts and for spots erroneously flagged by the software. Images of background and foreground intensities were produced for every sub-array by using bioinformatics tools. All spots or areas which did not represent a high quality signal were removed from analysis. Further quality controls were also performed  and the intensity values were background-corrected (index = Log2(foreground/background)).
False positive, “empty” spots removal and exclusion of low intensity signal spots
All spots showing a response on the buffer slides - and for this reason identified as possible false positive - were removed from the analysis, as well as all spots that did not show any signal (“empty”, with an index value ≤ −50) in the data acquisition process. Low response spots, with a signal below a computed cut off (μ + 2SD, where SD is the standard deviation of μ, the mean value of negative controls in the slides of each study group) were also removed.
the normalization process was performed using the simple linear model as described before [18,15,14,16,17]. The quality of the normalization was assessed by inspection of the normalized data plot in all the study groups.
Analysis and data mining
Data analysis was performed using two different statistical methods: (i) PAM (Prediction Analysis for Microarrays) , a predictive analysis which performs sample classification from peptide recognition data providing a list of significant peptides whose response level characterizes each diagnostic group. Compared to other differential recognition analysis methods, PAM is highly selective and allows the detailed examination of each time point in case of consecutive serum testing. This reveals only the peptide target with good predictive power associated with the differentiation of the patient group(s). This will result in a set of peptides constantly weakly recognized in one group and strongly in the other group. (ii) ‘Exclusive recognition analysis’ (ERA) of epitopes predicted by PAM. The latter approach identifies epitopes recognized in serum from individuals exclusively in one group but never in serum from any individual in a control reference group, e.g. in the current report the ‘reference’ individuals who received placebo (termed ‘exclusively recognized epitopes’, ERA). Strongly recognized peptides identified in each group were plotted according to index value and number of times they were recognized in the group of interest. Lastly, a 3D-graphical representation of the “reactome”  of B. pertussis proteins was computed for every group, by plotting mean index value for every peptide, as well as the protein and position on the respective amino acid-sequence of the protein. A similar 3D-plot was computed to compare the signals in two study groups, plotting the Δ value between the mean index values in the two groups (e.g. the Δ value for each individual reactivity, peptide by peptide in ‘test group’ as compared to the ‘reference’ group. The entire set of differences can be compiled in a 3D graphical representation).
All pre-processing and statistical analyses were performed customizing open-source packages of Bioconductor, R software [21,22]. In addition, to assess the statistical significance of the differences in the trends of recognition (defined as the sequence of the observations in 100%, 90% and 80% in serum from children in each group) with whooping cough group vs. all remaining groups, as well as the DT (control) group vs. the remaining groups, a Chi-square test for the goodness of fit was used.
Epitope comparison with published data
In order to relate our results to the epitopes which have been identified previously in the literature, we searched the B-cell Immune Epitope Database  (IEDB) site (http://www.immuneepitope.org/) and homologous sequences highlighted.
Differential recognition of Bp epitopes in children with whooping cough
Recognition frequency of target peptides spotted on the microarray slides, stratified per study group and by target proteins
B. pertussis protein
Pertussis toxin subunit 1 precursor
Pertussis toxin subunit 2 precursor
Pertussis toxin subunit 3 precursor
Pertussis toxin subunit 4 precursor
Pertussis toxin subunit 5 precursor
P.69A protein (pertactin)
Fim2 pilic subunit
Serotype 3 fimbrial subunit precursor
Tracheal colonization factor
Bifunctional hemolysin-adenylate cyclaseprecursor
Outer membrane porin protein precursor
Outer membrane porin protein OmpQ
GTP-binding elongation factor
Bordetella resistance to killing
Vag8 protein (Autotransporter)
Different IgG reactome and exclusive recognition of Bp epitopes in sera from infected children
Sequence of target epitopes exclusively recognized in serum from individuals either with the natural B. pertussis infection or after DTPa2 vaccination
B. pertussis protein
Children with whooping cough
Pertussis toxin subunit 4 precursor
P.69A protein (pertactin)
P.69A protein (pertactin)
Bifunctional hemolysin-adenylate cyclaseprecursor
Outer membrane porin protein precursor
Children who received the the DTPa2 vaccine
Bifunctional hemolysin-adenylate cyclaseprecursor
Tracheal colonization factor
Differential recognition of Bp epitopes identified by PAM segregates Bp vaccines
The exclusive epitope recognition analysis yields peptide targets that are unique for each test cohort. A different kind of analysis, PAM, identifies epitopes that are both always strongly recognized in the reference, and always weakly recognized in the ‘test’ group (or vice versa) in serum from each individual in the group. This allows predicting whether a reactivity pattern is associated to a certain defined endpoint (e.g. infection or vaccination, vaccination versus placebo). Alternatively, this method allows also comparing groups of individuals, i.e. individuals who received different kinds of Bp vaccines.
Peptide microarray analysis identifies previously described B-cell epitopes
Finally, we examined if already published Bp epitopes were captured by the peptide microarray matrix: sixty-five B. pertussis B-cell epitopes were retrieved from the IEDB (listed in the Additional file 2: Table S3, B-cell epitopes identified via online data repositories, in the online supplementary material, see also Figure S5, i.e. Plots of previously reported epitopes and epitopes using the peptide microarray approach, in the online data supplement), showing that the platform utilized in this report picks up already described B-cell epitopes. We identified Bp target peptides that were frequently recognized; these target peptides exhibited a variant amino acid sequence which occurs in natural Bp clinical isolates – the non-variant peptide epitopes were not recognized (see Additional file 2: Table S3, previously described epitopes, and S4, commonly recognized epitopes in serum from the respective patient groups, in the online data supplement).
Primary prevention remains the main intervention to limit pertussis occurrence and new transmission. The protective effect mediated by the Bp vaccine(s) appeared to vanish over time  and emergence of Bp strains carrying mutations of virulence factors has been reported 
Our study explored the immune response against Bp induced by natural infection and 3 different vaccines in children enrolled in a clinical trial conducted in Sweden : this trial showed that acellular vaccines, in particular DTPa5, ensured the best ratio between protection from whooping cough and acceptable rates of side effects.
We have been able to i) identify a high number of B-cell epitopes that have been described in the literature and the Bp epitope database with the peptide microarray technology described in this report (supplementary Table S5), ii) show robust differences between different vaccines concerning epitope recognition patterns (see Figures 5 and 6), and iii) picked up differences in IgG mutant Bp epitopes, i.e. that not the wildtype, yet the naturally occurring variant Bp epitope was recognized (Supplementary Figure S5). These results suggest that peptide microarrays provide a platform to visualize quantitative and qualitative differences in humoral recognition patterns at the epitope level. This may be relevant since genetic changes in Bp have been reported [24-28].
Serum from children with whooping cough displayed the broadest Bp epitope antibody recognition, with a certain number of peptides exclusively recognized in this group. Only serum analysis from individuals vaccinated with DTPwc showed a similar trend concerning the number of recognized Bp peptides, consistent with the fact that all the components of the bacterial wall are present in this vaccine preparation. The DTPa5 as well as the DTPa2 vaccine induced a significantly different humoral recognition pattern. The DTPa2 vaccine appeared to boost pre-existing Bp-reactive antibody responses as compared to the induction and expansion of new antibody reactivity pattern directed to new Bp target antigens.. This is reflected in stronger recognition of the proteins Bordetella resistance to killing (BrkA), Vag8 protein, Putative autotransporter (BapC). Conversely, humoral recognition of the filamentous hemagglutinin (FHA) appears to be induced by vaccination with the DTPwc vaccine as well as after natural infection with Bp.
The immune responses induced by Bp in the course of whooping cough after resolution is long-lasting and more protracted as compared to the immune response induced by vaccines; it may offer new potential targets to improve vaccine design for pertussis once the nature of the antibody reactivity mediating immune protection will be deciphered. The DTPa2 vaccine has a very good safety profile, yet its effect has been questioned in the past particularly in terms of protection and its duration . This could, in part, be explained by the fact that the DTPa2 vaccine acts by boosting a pre-existing ‘natural’ Bp recognition matrix, as compared to other vaccines which rather induced a shift in serum Bp epitope recognition patterns. Both DTPwc and DTPa5 showed a reactome similar to the Bp natural infection; future studies may address whether this would be related to increased protection induced by these vaccines compared to the DTPa2 .
There are at least four different, not mutually exclusive explanations concerning the spectrum of Bp target recognition induced by different vaccines: i) Vaccines could boost and modulate the recognition matrix for natural occurring and Bp specific antibodies directed against Bordetella spp, eliciting pre-existing humoral immune responses directed against Bp epitopes. This is consistent with the concept that ‘natural antibodies’ are a fundamental part of the immune system and play a crucial role in modulating the recognition (and response) to self and ‘non-self’ infectious antigens [29,30] ii) Children without Bp vaccination experience most likely the full-blown disease with the typical whooping cough presentation, yet some (non-vaccinated) individuals appear to experience limited disease, suggesting that ‘abortive’ cases or even immune protection may occur in the absence of vaccination [31,32]. Therefore, silent infection or colonization with other Bordetella species (which may also express certain virulence factors, e.g. B. parapertussis and B. bronchiseptica,, as well as B. trematum and B. holmesii) [33,34] could be responsible in the modulation of the immunological recognition matrix directed against Bp.
Most of the serum immune responses in the groups of our study were shared among the individuals in each group (with up to 11.9% of Bp peptides recognized in sera from 10/10 infants with whooping cough), yet we identified also ‘private’ humoral responses unique for each individuals (see Figure S2 in the online data supplement); iii) Potentially cross-reactive antibodies which target closely related hemagglutinin or fimbriae from other bacterial species may be responsible for different efficacy of the vaccines iv) Vaccination leads not only to epitope-specific immune responses directed against targets contained in the vaccine, yet to other molecular targets as well (epitope spreading). This concept is appreciated in other areas of medicine and contributes to clinical efficacy of some vaccines. For instance, antigen-spreading mediates vaccination-induced regression in human melanoma  and the impact of different (vaccine) adjutants on the antibody repertoire to target protective epitopes is appreciated in the development of humoral and cellular immune responses against influenza A [36,37].
If the human proteome is scanned as 5mer peptides, then up to 90% of the viral proteome may show similarity to the human proteome . This information is more easily accessible using peptide microarrays, since IgG recognition patterns are mapped using defined peptide targets that can be tested for amino acid composition similarities with related or unrelated protein targets.
We show here that not only the antibody titers directed against specific Bp targets, yet also the detailed recognition focus of vaccine components was different, even if the vaccines contained the same molecular components, supporting the notion that Bp vaccine composition impacts on the quality of antibody response [25,39-41].
Peptide-microarray-guided analysis may also help to decipher the phenomenon of ‘epitope suppression’  which has recently gained interest in Bp vaccine evaluation. Individuals primed with a Bp (one dose) whole cell vaccine exhibited decreased pertussis attack rates as compared to individuals primed with acellular vaccines [43,44]: differential target epitope focus associated with different vaccine formulation was evident in the PAM-analysis reported in the current study (see Figures 5 and 6, the detailed target epitope focus analysis is provided in the Additional file 2: Table S2).
Microarray analysis offered for the first time a comprehensive characterization of the immune response to Bp after natural infection in comparison to 3 vaccines. The report shows the potential of the high-content peptide microarray technique in infectious diseases, detecting epitopes by far more numerous and likely more immunogenic compared to the ones already reported in electronic databases. It also offers a new possibility to objectively decipher immune reactivity in clinically well-defined test groups undergoing vaccination strategies and allows to test for batch-to-batch consistency. Target recognition patterns in serum from individuals who experienced infection with Bp (and enjoyed protection for a longer period of time) could guide the development of Bp vaccines.
The authors wish to thank Vetenskapsrådet (Swedish research council) and Vinnova for supporting this study. We are indebted to A. Dewedar for helping in sample processing and data analysis.
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