Despite over 50 years of population-wide vaccination, whooping cough incidence is on the rise. AlthoughBordetella pertussis is considered the main causative agent of whooping cough in humans, Bordetella parapertussis infections are not uncommon. The widely used acellular whooping cough vaccines (aP) are comprised solely of B. pertussis antigens that hold little or no efficacy against B. parapertussis. Here, we ask how aP vaccination affects competitive interactions between Bordetella species within co-infected rodent hosts and thus the aP-driven strength and direction of in-host selection. We show that aP vaccination helped clear B. pertussis but resulted in an approximately 40-fold increase in B. parapertussis lung colony-forming units (CFUs). Such vaccine-mediated facilitation of B. parapertussis did not arise as a result of competitive release; B. parapertussis CFUs were higher in aP-relative to sham-vaccinated hosts regardless of whether infections were single or mixed. Further, we show that aP vaccination impedes host immunity against B. parapertussis—measured as reduced lung inflammatory and neutrophil responses. Thus, we conclude that aP vaccination interferes with the optimal clearance of B. parapertussis and enhances the performance of this pathogen. Our data raise the possibility that widespread aP vaccination can create hosts more susceptible to B. parapertussis infection.
Despite decades of worldwide pertussis vaccination, whooping cough is re-emerging in highly vaccinated countries (CDC 2002; Celentano et al. 2005). A rise in non-vaccine alleles coincident with widespread vaccination has been documented for Bordetella pertussis (Elomaa et al. 2005; Van Amersfoorth et al. 2005; Van Gent et al. 2009) leading some authors to propose that vaccine-driven epitope-evolution in B. pertussis is one factor—among several others (Berbers et al. 2009)—that may contribute to whooping cough re-emergence in humans (Mooi et al. 2001). However, it is not clear how Bordetella parapertussis—the other major aetiological agent of human whooping cough—might respond to the selective pressure exerted by large-scale pertussis vaccination. Here, we postulate that the widespread and long-term use of acellular subunit pertussis vaccines creates hosts that are more favourable for B. parapertussis.
All commercial whooping cough vaccines currently contain either killed whole cells or purified antigens ofB. pertussis—herein referred to as whole cell (wP) and acellular vaccines (aP), respectively. Currently, aP vaccines are largely favoured over their wP predecessors owing to their reduced reactogenicity (Andersonet al. 1988). Although aP vaccines are very effective at reducing the incidence of B. pertussis infection (Mattoo & Cherry 2005), they hold little or no efficacy against B. parapertussis (Stehr et al. 1998; Willemset al. 1998; Liese et al. 2003; David et al. 2004). In fact, B. parapertussis prevalence is predicted to increase slightly in response to vaccines that are less protective against B. parapertussis than natural B. pertussis infection (Restif et al. 2008). Thus, analogous to the serotype specificity observed for conjugate vaccines against other infectious diseases and the serotype replacement associated with their use (Obaro et al. 1996; Lipsitch 1997), we hypothesize that the prolonged and widespread use of B. pertussis-specific aP vaccines has the potential to increase carriage of species not included in the vaccine, namely B. parapertussis.
The rationale to design and employ vaccines that target only B. pertussis stems from the assumption that B. parapertussis infections are not widely prevalent. Indeed, the vast majority of whooping cough studies do not attempt to identify B. parapertussis because differential diagnosis does not affect clinical management and this probably leads to under-reporting. However, when differential diagnosis has been carried out, B. parapertussis was found to comprise between 2 and 36 per cent of cases (Watanabe & Nagai 2004) and, in one study, to constitute the major aetiological agent (Borska & Simkovicova 1972). Both mixed and sequential infections of B. pertussis and B. parapertussis have been reported in epidemiological studies (Mertsola 1985; Iwata et al. 1991; He et al. 1998; Mastrantonio et al. 1998; Stehr et al. 1998; Bergfors et al. 1999), showing that B. pertussis and B. parapertussis co-circulate in the same populations and sometimes the same hosts.
Some aP vaccine efficacy studies report a significantly higher proportion of B. parapertussis relative to B. pertussis in aP-vaccinated compared with unvaccinated individuals (Bergfors et al. 1999; Liese et al. 2003). These data are consistent with the hypothesis that B. parapertussis gains a selective advantage under aP vaccination. We can envisage at least three possible mechanisms by which aP vaccination could generate this selective advantage, all of which are based on the observation that aP vaccination confers less protection against B. parapertussis than the immunity induced by natural B. pertussis infection or wP vaccination.
First, aP vaccination could drive competitive release within individual hosts (Grech et al. 2008; Read & Mackinnon 2008). The transmission success of a given pathogen genotype depends on its intrinsic fitness and competitive ability (Read & Taylor 2001). Theory has predicted that B. pertussis must have a competitive advantage over B. parapertussis in unvaccinated co-infected hosts (Restif et al. 2008). However, aP vaccination can give B. parapertussis two potential fitness advantages; first, it can better survive aP vaccination than B. pertussis (Stehr et al. 1998; Willems et al. 1998; Liese et al. 2003; David et al. 2004) and second, by removing B. pertussis competitors, it could open up ecological space for B. parapertussis, which can greatly enhance the rate of spread of non-vaccine B. parapertussis (competitive release hypothesis; Lipsitch 1997; Hastings & D’Alessandro 2000). A second possibility is that by focusing immune responses on B. pertussis, aP vaccination interferes with an optimal immune response against B. parapertussis, resulting in slower clearance or enhanced establishment of B. parapertussis (enemy release hypothesis (ERH)). ERH is a term used widely in plant ecology when a plant species experiences a decrease in regulation by ‘natural enemies’ and rapidly increases in distribution and abundance (Mitchell & Power 2003). Such natural enemies might constitute herbivores in the case of plant ecology and, in pathogen biology, host immunity. Results from one aP vaccine efficacy study examining B. parapertussisin mice are consistent with an aP-driven enhancement of B. parapertussis infection (David et al. 2004), but is unclear whether a lack of immune regulation was driving this enhancement. A third possibility—but one not easily testable empirically for ethical reasons—is that aP vaccination could increase the number of humans susceptible to B. parapertussis by reducing levels of cross-immunity that would have otherwise been generated by natural B. pertussis infections or wP vaccination. Under this scenario, vaccination is in effect creating new ecological opportunities for B. parapertussis (the vacant niche filling hypothesis).
Here, we used a rodent model of B. pertussis and B. parapertussis infection to investigate the competitive ERH. By vaccinating laboratory mice with a commercial aP vaccine (which selectively targets B. pertussisand not B. parapertussis) and challenging them with single- or mixed-species infections (table S1, electronic supplementary material), the level of protection and immune stimulation was estimated over time in terms of changes in lung colony-forming units (CFUs), cytokine milieu, neutrophil recruitment and pathogen-specific antibody responses. If B. parapertussis is competitively suppressed by B. pertussisinfection, B. parapertussis lung CFU will be lower in mixed relative to single infections (tested with the term ‘infection type’, a two-level factor describing the number of Bordetella species present in an infection; single or mixed). Following from this, competitive release of B. parapertussis would present as a significant interaction between infection type and ‘vaccination’—a two-level factor describing the vaccination regime administered, sham or aP, and the infection type. If enemy release is occurring, we expect B. parapertussis CFUs to be higher in aP-vaccinated relative to sham-vaccinated hosts (tested with the term vaccination), regardless of whether infections were alone or in a mixture (which would present as a significant main effect of vaccination and a non-significant interaction between vaccination and infection type). Evidence that aP vaccination interferes with an optimal host immune response against B. parapertussis would further support the ERH. Our results support the enemy release model: aP vaccination interferes with the optimal clearance of B. parapertussis and enhances the performance of this pathogen.
2. Material and methods
(a) Bacteria strains and growth conditions
Bordetella pertussis 1740 is a derivative of Tohama I (Kasuga et al. 1954), rendered kanamycin resistant by the chromosomal insertion of pSS4266 (Goebel et al. 2008) and was a kind gift from Dr Scott Stibitz (USDA). Bordetella parapertussis 12822 was isolated from German clinical trials (Heininger et al. 2002) and 12822G is a gentamicin-resistant derivative of the parent strain (Wolfe et al. 2005). Bordetellae were maintained on Bordet-Gengou (BG) agar (Difco) containing 10 per cent defibrinated sheep blood (Hema Resources) at 37°C for approximately 72 h. Supplementing BG plates with kanamycin or gentamicin (50 and 20 µg ml−1, respectively; Sigma Aldrich) allowed differentiation between bacteria in mixed infections. For experimental inocula, liquid culture bacteria were grown overnight at 37°C and shaken to mid-log phase (optical density at 600 nm of approx. 0.3) in Stainer-Scholte broth.
(b) Hosts, vaccination and inoculation
Four- to six-week-old female C57BL/6 mice (Jackson Laboratories) were maintained in specific pathogen-free rooms at Pennsylvania State University and were handled in accordance with Institutional Animal Care and Use Committee guidelines. In two experiments, a total of 200 mice were divided into eight treatment groups. Half of all mice received two 50 µl subcutaneous injections (on days 0 and 14) of the commercial Adacel vaccine (referred to as aP; Sanofi Pasteur) at one-fifth the human dose, whereas the other half were sham vaccinated sterile phosphate buffered saline (PBS) and both treatments were administered with Imject Alum adjuvant (Thermo Scientific). Using this vaccination protocol, vaccine efficacy in human clinical trials was shown to correlate with bacterial clearance in a murine model of B. pertussis (Mills et al. 1998; Guiso et al. 1999). Adacel vaccines are provided as combined tetanus–diphtheria–pertussis formulation adsorbed to alum and contain the following five B. pertussis antigens: 5 µg ml−1 of detoxified pertussis toxin, 10 µg ml−1 filamentous haemagglutinin, 6 µg of pertactin and 10 µg of fimbriae types 2 and 3.
In both vaccinated and sham-vaccinated groups, 27 mice were each infected with B. pertussis alone, B. parapertussis alone or a mixture of both, and 21 were sham infected with sterile PBS (table S1, electronic supplementary material). Mice were challenged intranasally with 5 × 106 CFU three weeks after the second vaccination (day 35), as described (Harvill et al. 1999). For mixed infections, the 50 µl inocula contained 5 × 106 CFU of each of B. pertussis and B. parapertussis. The same dose of each bacterium in mixed and single infections was used as we wanted to compare the dynamics of each bacterium on its own versus in mixed infections. On each day of sacrifice (table S1, electronic supplementary material; experiment 1: days 0, 3, 7, 14 and 35 post-infection (p.i.) and experiment 2: days 0, 3 and 7 p.i.), three to four mice per group were sacrificed in experiments 1 and 2, respectively. In both experiments, lungs were aseptically removed and homogenized in 1 ml of sterile PBS. Serial dilutions of organ homogenate were plated on BG agar plates containing the relevant antibiotics and cells were incubated for 3–5 days at 37°C to quantify the number of viable bacteria. In experiment 1, blood was collected on each day of sacrifice for the assessment of Bordetella-specific serum antibodies.
(c) Lung cytokines, neutrophil numbers and antibody enzyme linked immunosorbent assays (ELISAs)
In experiment 1, levels of the lung cytokines interleukin (IL)-4, IL-5, interferon gamma (IFN-γ) and granulocyte macrophage-colony stimulating factor (GM-CSF) were quantified using a flow cytometric cytokine assay, according to the manufacturer’s instructions (Bio-Plex Mouse Cytokine TH1/TH2 Panel Cytokine Assay and a Bio-Plex cytokine reagent kit, Bio-Rad).
In experiment 2, lung leukocyte numbers were quantified by performing lung perfusions on days 0, 3 and 7 p.i. Briefly, lungs were perfused with sterile PBS on day of sacrifice to remove red blood cells before homogenizing through sterile cell strainers (BD Biosciences). Homogenates were laid on a Histopaque gradient 1119 (Sigma Aldrich), centrifuged at 1500 g for 30 min and the leukocyte layer collected and counted on a haemocytometer at ×40 magnification. Aliquots of cells were stained with fluorescein labelled antibodies (FITC)-labelled anti-Ly-6G to detect neutrophils (eBioscience). The percentage of FITC-positive cells was multiplied by the total number of leukocytes to calculate neutrophil numbers.
Bacteria were grown overnight (optical density of 0.7 at 600 nm), diluted in carbonate buffer and 200 µl added to each well of a 96-well plate. Serum from experiment 1 was added to the first row of coated 96-well plates at a 1 : 50 dilution and serially diluted across the plates to a final dilution of 1 : 102 400. Incubation, wash and development steps were carried out as detailed (Wolfe et al. 2005). Total immunoglobulin (Ig) titre was quantified using biotin-conjugated anti-mouse Ig (Southern Biotechnology Associates) and peroxidase-conjugated streptavidin (BD Pharmingen). Results were reported as endpoint titres.
(d) Statistical analyses
All analyses were performed in R v. 2.7.0 (http://www.R-project.org) using generalized linear models (GLM; Crawley 2007; R 2008). Analyses focused on 200 mice, with experimental groups as detailed above and in table S1, electronic supplementary material. We assumed lognormal errors in CFU, cytokine and antibody titres and carried out the analysis on the log10 transformed data, using least squares with normal errors and the identity link. Data from duplicate bioassay and triplicate antibody enzyme linked immunosorbent assay (ELISA) plate wells were averaged and the respective titres induced by naive animals were subtracted from experimental animals before being log10(n + 1) transformed to satisfy homogeneity-of-variance and normality-of-error assumptions of models used.
CFU, lung cytokine and antibody data were analysed from days 3 to 35 p.i. inclusive in order to capture the full post-peak dynamics of infection. Main effects were vaccination (aP versus sham vaccinated), infection type (single versus mixed infection) and day p.i. (fitted as a categorical variable). The main effects of infection, vaccination and the infection by vaccination interaction terms explicitly test the main hypothesis of this study. To control for the dynamic kinetics of Bordetella infection, the main effect of day—as well as all two-way interactions between day and vaccination or infection type—was included in all analyses. In no cases were any of the three-way interactions significant and so they are not reported. Qualitative differences owing to infection type and vaccination were strong and consistent across experimental blocks and quantitative differences were controlled for by including experimental block as a factor in all analyses. Maximal models were first fit to the data and minimal models reached by removing non-significant terms (p > 0.05), beginning with the highest level interaction. Reported parameter estimates were taken from the relevant minimal models.
(a) Vaccine-mediated interactions and mixed infection
As expected, aP vaccination significantly reduced the CFU of B. pertussis (figure 1a,b; CFU days 3–35 inclusive, vaccination (acellular or sham): F1,72 = 145.9, p < 0.0001; vaccination × day: F3,72 = 6.5, p = 0.001). The average bacterial abundance produced throughout the infection was approximately 700-fold lower in aP-vaccinated relative to sham-vaccinated hosts. By contrast, aP vaccination significantly increased B. parapertussis CFU (figure 1c,d; CFU days 3–35 inclusive, vaccination: F1,72 = 16.9, p < 0.0001; vaccination × day: F3,72 = 5.5, p = 0.002). The average bacterial abundance produced throughout infection was approximately 40-fold higher in aP-vaccinated relative to sham-vaccinated hosts.
Bacterial lung CFUs. (a) Timeline of B. pertussis and (c) B. parapertussis in single and mixed infections of aP- or sham-vaccinated hosts. Average CFU between days 3–35 p.i. (least-squares mean ± s.e.m.) from (b) GLMs of B. pertussis CFU …
We found no evidence to support within-host competitive suppression of either pathogen species by the other. For B. pertussis, the vaccine-driven decrease in bacterial abundance was observed independent of whether infections were alone or in a mixture with B. parapertussis (figure 1a,b; CFU days 3–35 inclusive, infection type (mixed versus single) and infection type × vaccination, infection type × day, all p > 0.05) and was of similar magnitude in both experimental blocks (block × vaccination: p > 0.05). Likewise, the increase in B. parapertussis CFU was unaffected by the presence of B. pertussis (figure 1c,d; CFU days 3–35 inclusive, infection type, infection type × vaccination, infection type × day and block × vaccination, allp > 0.05). Thus, we found no support for the competitive release hypothesis: there was no competition and hence no expansion of B. parapertussis when B. pertussis was selectively suppressed by aP vaccination.
Consistent with this absence of competition, mixed infections had an approximately twofold higher average CFU relative to single infections (CFU days 3–35 inclusive, infection type: B. pertussis, F1,75 = 67.9, p < 0.0001; B. parapertussis, F1,75 = 39.4, p < 0.0001, respectively), implying that there is no constrained ‘niche space’ over which the two species were competing. Thus, aP vaccination enhanced B. parapertussis CFUs in the lung, reversing the dominance from B. pertussis to B. parapertussis independent of the multiplicity of infection, consistent with the ERH.
(b) Lung cytokines and neutrophil recruitment
The lung immune response was skewed from a TH1 towards a predominantly TH2 response by aP vaccination. Specifically, aP-vaccinated mice produced significantly lower IFN-γ and higher lung IL-5 and IL-4 levels—a cytokine profile characteristic of TH2 cells—relative to sham-vaccinated mice (figure 2a–c; (cytokine) between days 3 and 35, inclusive: IFN-γ vaccination, F1,64=23.75, p < 0.0001 and vaccination × day, F3,64=8.9, p < 0.0001; IL-5 vaccination, F1,67 = 14.5, p < 0.0001 and vaccination × day, p > 0.05; IL-4 vaccination, F1,70 = 6.0, p = 0.02 and vaccination × day, p > 0.05) and this was true for both Bordetellaspecies (infection type, infection type × vaccination and infection type × day terms, all p > 0.05). In addition, lung GM-CSF levels were significantly reduced from day 3 p.i. onwards in aP-vaccinated hosts, regardless of the Bordetella species or multiplicity of infection (figure 2d; (cytokine) between days 3 and 35 inclusive: vaccination, F1,64 = 20.39, p < 0.0001; vaccination × day, F3,64=3.0, p = 0.03; infection type, infection type × vaccination and infection type × day terms, all p > 0.05).
Lung cytokine profiles. Plots show (a) timeline of IFN-γ, (b) IL-5, (c) IL-4 and (d) GM-CSF levels induced in lung homogenate during single- and mixed-B. pertussis and B. parapertussis infections in aP- or sham-vaccinated hosts. The values from …
Although the number of neutrophils recruited to the lungs early in infection was significantly lower in aP- relative to sham-vaccinated hosts, the extent of this aP-driven reduction in neutrophils depended onBordetella species; aP-vaccinated hosts infected with B. parapertussis (either as a single or mixed infection) had significantly lower neutrophil numbers compared with B. pertussis-infected individuals (figure 3; lung neutrophil numbers on days 3–7: vaccination, F1,58 = 4.2, p = 0.04; vaccination × day, p > 0.05; infection type, F2,58 = 0.46, p = 0.6; infection type × day, F2,58 = 3.1, p = 0.02; infection type × vaccination, F2,58 = 3.8, p = 0.03).
Lung neutrophil recruitment. Plots show neutrophil numbers in the lung on days 3 and 7 p.i. broken down by infection. Each plotted point represents the mean of 12 mice ± s.e.m. Filled (sham) and open (aP) triangles represent B. parapertussis; …
(c) Pathogen-specific antibody response
Acellular vaccination enabled both B. pertussis– and B. parapertussis-infected hosts to mount more rapid anti-B. pertussis- and anti-B. parapertussis-specific Ig responses, respectively, relative to their sham-vaccinated counterparts (figure 4a,b; B. pertussis: vaccination, F1,37 = 23.3, p < 0.001; vaccination × day,F3,37 = 4.9, p = 0.006; figure 4c,d; B. parapertussis: vaccination, F1,37 = 24.2, p < 0.0001; vaccination × day, F3,37 = 3.9, p = 0.02, respectively). The extent to which aP vaccination affected the anamnestic responses depended on whether the infection was single or a mixture (figure 4a–d; B. pertussis: infection type, F1,37 = 0.8, p = 0.4; infection type × vaccination, F1,37 = 4.6, p = 0.03; B. parapertussis, infection type, F1,37 = 0.08, p = 0.8; infection type × vaccination, F1,37 = 4.7, p = 0.04).
Bordetella species-specific serum Ig titres. Timeline showing titres of (a) anti-B. pertussis total Ig and (c) anti-B. parapertussis total Ig during single and mixed B. pertussis and B. parapertussis infections of sham or vaccinated hosts. (a and c) Filled …
Here we show that aP vaccination accelerated the clearance of B. pertussis from the lower respiratory tract (LRT) of mice (figure 1a,b), but delayed B. parapertussis clearance, resulting in approximately 40-fold higher total B. parapertussis lung CFUs (figure 1c,d). Importantly, no evidence to support competitive interactions between B. pertussis and B. parapertussis was found in either sham- or aP-vaccinated co-infected hosts (figure 1a–d). An aP vaccine-driven reduction in inflammatory cytokine responses (figure 2) as well as neutrophil recruitment to the lung in response to B. parapertussis infection (figure 3)—two key players in the clearance of this pathogen (Kirimanjeswara et al. 2005; Mann et al. 2005; Wolfe et al. 2009)—correlated with delayed B. parapertussis clearance. In addition, antibody responses in vaccinated B. parapertussis-infected hosts, although robust, were likely to have reduced efficacy relative to non-vaccinated hosts owing to species differences in prominent surface molecules preventing immune cross-protection (Wolfe et al. 2007; Zhang et al. 2009a,b). Thus aP vaccination, by priming the host response against B. pertussis clearance, confers an advantage to B. parapertussis by interfering with optimal immune clearance and resulting in increased lung CFUs, consistent with the ERH outlined in §1 (Mitchell & Power 2003).
Bordetella parapertussis and B. pertussis have been thought to compete directly with one another as they exploit the same respiratory tract niche (Bjørnstad & Harvill 2005). However, we found no evidence of within-host competition between B. parapertussis and B. pertussis in our study: CFUs of each species appeared to be unaffected by the presence of the other (figure 1a–d). The lack of competition probably arose as total infection densities were not constrained in the lung: infection with two species resulted in total pathogen densities twice that of single-species infections. Indeed, by colonizing discrete areas in the LRT, these distinct infections may avoid direct interaction. Another possibility is that by focusing solely on the LRT, we failed to capture within-host competition between B. parapertussis and B. pertussis in the upper respiratory tract (URT). Bordetella infection is initiated by the attachment of organisms to epithelial cell cilia of the URT, a respiratory area that is thought to act as an important reservoir of Bordetellainfection (Mattoo & Cherry 2005). Experiments examining the localization of distinct bacterial populations in both the URT and LRT, as well as transmission of bacteria from the respiratory tract (which can be carried out experimentally in rat or rabbit models of bordetellosis), would increase our understanding of colonization and shedding processes respectively, and how these may vary with vaccination status orBordetella species.
What mechanisms are behind the ‘enemy release’ of B. parapertussis under aP vaccination and why does wP vaccination or prior exposure to B. pertussis not drive similar increases in B. parapertussis CFU (Wolfeet al. 2007; Zhang et al. 2009b)? First, robust TH1 inflammatory responses and neutrophil recruitment to the LRT are required for optimal anamnestic responses against B. parapertussis (Kirimanjeswara et al. 2005; Mann et al. 2005; Wolfe et al. 2005, 2009). Here, we show that aP vaccination skews the host immune response towards a TH2 response (Barnard et al. 1996; Ryan et al. 1997) and it is likely that this lack of inflammatory help—reduced lung inflammatory responses and neutrophil recruitment—enables B. parapertussis to evade rapid antibody-mediated clearance in our study (Wolfe et al. 2009). Second, omission of the critical protective O-antigen from aP vaccine preparations is also likely to reduce aP vaccine efficacy against B. parapertussis (Zhang et al. 2009a) and could contribute towards enhanced infection. Third, aP vaccination may have the potential to provoke immune interference in the form of original antigenic sin. Of those B. pertussis antigens contained in the aP vaccine expressed by B. parapertussis, antigenic differences exist between the Bordetella species and so individuals exposed to a B. parapertussis antigen similar, but not identical to one encountered previously, may induce an immune response to the latter antigen directed against the first (Francis 1953; Webster 1966; Klenerman & Zinkernagel 1998). Thus, subunit vaccines with limited epitopes—such as the aP vaccine—may have the potential to prevent appropriate immune responses against challenging B. parapertussis bacteria whose epitopes are divergent from those of the vaccine variant and lead to sub-optimal clearance and perhaps enhanced infection.
Importantly, following the effects of aP vaccination on infection dynamics over time allowed us to resolve previously conflicting results concerning the effect of aP on B. parapertussis (David et al. 2004; Zhang et al. 2009b). Specifically, we show that the effect of aP vaccination on B. parapertussis infection varied temporally—aP vaccination did not affect B. parapertussis lung CFU on day 3 p.i. consistent with Zhang et al. (2009b), but enhanced CFU on day 7 p.i. consistent with David et al. (2004) (figure 1a–d)—which resolves these previously conflicting studies and highlights the importance of tracking dynamics throughout infection in order to capture full effects of the treatment of interest. It is possible that these findings may be relevant only to the specific strains we have examined and further studies should be carried out to determine if our results hold across B. pertussis and B. parapertussis strains.
As always, it is important to be cautious about extrapolating from animal models to humans. The dynamics of B. pertussis and B. parapertussis infection in rodent hosts shares many similarities with human infection, but like all experimental models, differs from the human situation in a number of key ways (Elahi et al. 2007). However, the relative efficacies of pertussis vaccines in the rodent model correspond to those obtained in clinical trials (Mills et al. 1998; Guiso et al. 1999), and we note that epidemiological evidence in human whooping cough infections is consistent with an enhancement effect for B. parapertussis (Bergfors et al. 1999; Liese et al. 2003). Directly proving aP vaccination puts treated people at risk of acquiring B. parapertussis is very difficult, but we hope our study highlights the need for more thorough B. parapertussis epidemiological data and encourages further work in this neglected area. If our experiments are capturing the phenomenology of what is happening under aP vaccination in humans, it may be important to consider the introduction of vaccines that better protect against both bordetellae; for example, live attenuated B. pertussis nasal vaccines (Mielcarek et al. 2006), wP vaccines containing bothB. pertussis and B. parapertussis (Burianova-Vysoka et al. 1970), or supplementation of aP vaccines withB. parapertussis protective antigens (Zhang et al. 2009a). An enhanced understanding of the evolutionary consequences of widespread aP vaccination is needed in order to optimize the next generation of vaccination strategies and fully reap the benefits of this powerful medical intervention.
All procedures were carried out in accordance with Institutional Animal Care and Use Committee guidelines.
We are grateful to D. Bann for help with ELISAs, for valuable discussions with D. Wolfe, A. Buboltz, X. Zhang, J. Thakar and S. Perkins and two anonymous reviewers for comments that greatly improved the manuscript. This research was supported by a Center for Infectious Disease Dynamics Postdoctoral Fellowship (GHL), NSF grant EF-0520468 as part of the joint NSF-NIH Ecology of Infectious Disease program (PJH) and NIH grant GM083113 (ETH) and benefitted from the RAPIDD working group on zoonoses and emerging disease threats.
- Anderson E. L., Belshe R. B., Bartram J. 1988Differences in reactogenicity and antigenicity of acellular and standard pertussis vaccines combined with diphtheria and tetanus in infants. J. Infect. Dis. 157, 731–737 [PubMed]
- Barnard A., Mahon B. P., Watkins J., Redhead K., Mills K. H. 1996Th1/Th2 cell dichotomy in acquired immunity to Bordetella pertussis: variables in the in vivo priming and in vitro cytokine detection techniques affect the classification of T-cell subsets as Th1, Th2 or Th0. Immunology 87, 372–380 (doi:10.1046/j.1365-2567.1996.497560.x) [PMC free article] [PubMed]
- Berbers G. A., de Greeff S. C., Mooi F. R. 2009Improving pertussis vaccination. Hum. Vaccine 5, 497–503[PubMed]
- Bergfors E., Trollfors B., Taranger J., Lagergard T., Sundh V., Zackrisson G. 1999Parapertussis and pertussis: differences and similarities in incidence, clinical course, and antibody responses. Int. J. Infect. Dis. 3, 140–146 (doi:10.1016/S1201-9712(99)90035-8) [PubMed]
- Bjørnstad O. N., Harvill E. T. 2005Evolution and emergence of Bordetella in humans. Trends Microbiol.13, 355–359 (doi:10.1016/j.tim.2005.06.007) [PubMed]
- Borska K., Simkovicova M. 1972Studies on the circulation of Bordetella pertussis and Bordetella parapertussis in populations of children. J. Hyg. Epidemiol. Microbiol. Immunol. 16, 159–172[PubMed]
- Burianova-Vysoka B., Burian V., Kriz B., Pellar T., Hellerova J., Radkovsky J. 1970Experiences with a new combined vaccine against B. parapertussis, diphtheria, tetanus and B. pertussis employed in a field trial in Prague 10. J. Hyg. Epidemiol. Microbiol. Immunol. 14, 481–487 [PubMed]
- CDC 2002From the Centers for Disease Control and Prevention. Pertussis–United States, 1997–2000. J. Am. Med. Assoc. 287, 977–979 (doi:10.1001/jama.287.8.977) [PubMed]
- Celentano L. P., Massari M., Paramatti D., Salmaso S., Tozzi A. E. 2005Resurgence of pertussis in Europe.Pediatr. Infect. Dis. J. 24, 761–765 (doi:10.1097/01.inf.0000177282.53500.77) [PubMed]
- Crawley M. J. 2007The R Book Bognor Regis, UK: John Wiley & Sons
- David S., Van Furth R., Mooi F. R. 2004Efficacies of whole cell and acellular pertussis vaccines againstBordetella parapertussis in a mouse model. Vaccine 22, 1892–1898 (doi:10.1016/j.vaccine.2003.11.005) [PubMed]
- Elahi S., Holmstrom J., Gerdts V. 2007The benefits of using diverse animal models for studying pertussis.Trends Microbiol. 15, 462–468 (doi:10.1016/j.tim.2007.09.003) [PubMed]
- Elomaa A., Advani A., Donnelly D., Antila M., Mertsola J., Hallander H., He Q. 2005Strain variation among Bordetella pertussis isolates in Finland, where the whole-cell pertussis vaccine has been used for 50 years. J. Clin. Microbiol. 43, 3681–3687 (doi:10.1128/JCM.43.8.3681-3687.2005)[PMC free article] [PubMed]
- Francis T., Jr 1953Influenza: the new acquaintance. Ann. Intern. Med. 39, 203–221 [PubMed]
- Goebel E. M., Wolfe D. N., Elder K., Stibitz S., Harvill E. T. 2008O antigen protects Bordetella parapertussis from complement. Infect. Immun. 76, 1774–1780 (doi:10.1128/IAI.01629-07)[PMC free article] [PubMed]
- Grech K., Chan B. H., Anders R. F., Read A. F. 2008The impact of immunization on competition withinPlasmodium infections. Evolution 62, 2359–2371 (doi:10.1111/j.1558-5646.2008.00438.x) [PubMed]
- Guiso N., Capiau C., Carletti G., Poolman J., Hauser P. 1999Intranasal murine model of Bordetella pertussis infection. I. Prediction of protection in human infants by acellular vaccines. Vaccine 17, 2366–2376 (doi:10.1016/S0264-410X(99)00037-7) [PubMed]
- Harvill E. T., Cotter P. A., Yuk M. H., Miller J. F. 1999Probing the function of Bordetella bronchisepticaadenylate cyclase toxin by manipulating host immunity. Infect. Immun. 67, 1493–1500[PMC free article] [PubMed]
- Hastings I. M., D’Alessandro U. 2000Modelling a predictable disaster: the rise and spread of drug-resistant malaria. Parasitol. Today 16, 340–347 (doi:10.1016/S0169-4758(00)01707-5) [PubMed]
- He Q., Viljanen M. K., Arvilommi H., Aittanen B., Mertsola J. 1998Whooping cough caused by Bordetella pertussis and Bordetella parapertussis in an immunized population. J. Am. Med. Assoc. 280, 635–637 (doi:10.1001/jama.280.7.635) [PubMed]
- Heininger U., Cotter P. A., Fescemyer H. W., Martinez de Tejada G., Yuk M. H., Miller J. F., Harvill E. T. 2002Comparative phenotypic analysis of the Bordetella parapertussis isolate chosen for genomic sequencing. Infect. Immun. 70, 3777–3784 (doi:10.1128/IAI.70.7.3777-3784.2002) [PMC free article][PubMed]
- Iwata S., et al. 1991Mixed outbreak of Bordetella pertussis and Bordetella parapertussis in an apartment house. Dev. Biol. Stand. 73, 333–341 [PubMed]
- Kasuga T., Nakase Y., Ukishima K., Takatsu K. 1954Studies on Haemophilus pertussis. V. Relation between the phase of bacilli and the progress of the whooping-cough. Kitasato Arch. Exp. Med. 27, 57–62 [PubMed]
- Kirimanjeswara G. S., Mann P. B., Pilione M., Kennett M. J., Harvill E. T. 2005The complex mechanism of antibody-mediated clearance of Bordetella from the lungs requires TLR4. J. Immunol. 175, 7504–7511 [PubMed]
- Klenerman P., Zinkernagel R. M. 1998Original antigenic sin impairs cytotoxic T lymphocyte responses to viruses bearing variant epitopes. Nature 394, 482–485 (doi:10.1038/28860) [PubMed]
- Liese J. G., Renner C., Stojanov S., Belohradsky B. H. 2003Clinical and epidemiological picture of B. pertussis and B. parapertussis infections after introduction of acellular pertussis vaccines. Arch. Dis. Child 88, 684–687 (doi:10.1136/adc.88.8.684) [PMC free article] [PubMed]
- Lipsitch M. 1997Vaccination against colonizing bacteria with multiple serotypes. Proc. Natl Acad. Sci. USA 94, 6571–6576 (doi:10.1073/pnas.94.12.6571) [PMC free article] [PubMed]
- Mann P. B., Wolfe D., Latz E., Golenbock D., Preston A., Harvill E. T. 2005Comparative toll-like receptor 4-mediated innate host defense to Bordetella infection. Infect. Immun. 73, 8144–8152 (doi:10.1128/IAI.73.12.8144-8152.2005) [PMC free article] [PubMed]
- Mastrantonio P., Stefanelli P., Giuliano M., Herrera Rojas Y., Ciofi degli Atti M., Anemona A., Tozzi A. E. 1998Bordetella parapertussis infection in children: epidemiology, clinical symptoms, and molecular characteristics of isolates. J. Clin. Microbiol. 36, 999–1002 [PMC free article] [PubMed]
- Mattoo S., Cherry J. D. 2005Molecular pathogenesis, epidemiology, and clinical manifestations of respiratory infections due to Bordetella pertussis and other Bordetella subspecies. Clin. Microbiol. Rev.18, 326–382 (doi:10.1128/CMR.18.2.326-382.005) [PMC free article] [PubMed]
- Mertsola J. 1985Mixed outbreak of Bordetella pertussis and Bordetella parapertussis infection in Finland.Eur. J. Clin. Microbiol. 4, 123–128 (doi:10.1007/BF02013576) [PubMed]
- Mielcarek N., et al. 2006Live attenuated B. pertussis as a single-dose nasal vaccine against whooping cough. PLoS Pathog. 2, e65 (doi:10.1371/journal.ppat.0020065) [PMC free article] [PubMed]
- Mills K. H., Ryan M., Ryan E., Mahon B. P. 1998A murine model in which protection correlates with pertussis vaccine efficacy in children reveals complementary roles for humoral and cell-mediated immunity in protection against Bordetella pertussis. Infect. Immun. 66, 594–602 [PMC free article][PubMed]
- Mitchell C. E., Power A. G. 2003Release of invasive plants from fungal and viral pathogens. Nature 421, 625–627 (doi:10.1038/nature01317) [PubMed]
- Mooi F. R., Van Loo I. H., King A. J. 2001Adaptation of Bordetella pertussis to vaccination: a cause for its reemergence? Emerg. Infect. Dis. 7, 526–528 (doi:10.3201/eid0703.010308) [PMC free article][PubMed]
- Obaro S. K., Adegbola R. A., Banya W. A., Greenwood B. M. 1996Carriage of pneumococci after pneumococcal vaccination. Lancet 348, 271–272 (doi:10.1016/S0140-6736(05)65585-7) [PubMed]
- R 2008R Development Core Team. R: a language and environment for statistical computing Vienna, Austria: R Foundation for Statistical Computing
- Read A. F., Mackinnon M. J. 2008Pathogen evolution in a vaccinated world. In Evolution in health and disease, 2nd edn (eds Stearns S. C., Koella J., editors. ), pp. 139–152 Oxford, UK: Oxford University Press
- Read A. F., Taylor L. H. 2001The ecology of genetically diverse infections. Science 292, 1099–1102 (doi:10.1126/science.1059410) [PubMed]
- Restif O., Wolfe D. N., Goebel E. M., Bjørnstad O. N., Harvill E. T. 2008Of mice and men: asymmetric interactions between Bordetella pathogen species. Parasitology 135, 1517–1529 (doi:10.1017/S0031182008000279) [PubMed]
- Ryan M., Gothefors L., Storsaeter J., Mills K. H. 1997Bordetella pertussis-specific Th1/Th2 cells generated following respiratory infection or immunization with an acellular vaccine: comparison of the T cell cytokine profiles in infants and mice. Dev. Biol. Stand. 89, 297–305 [PubMed]
- Stehr K., et al. 1998A comparative efficacy trial in Germany in infants who received either the Lederle/Takeda acellular pertussis component DTP (DTaP) vaccine, the Lederle whole-cell component DTP vaccine, or DT vaccine. Pediatrics 101, 1–11 (doi:10.1542/peds.101.1.1) [PubMed]
- Van Amersfoorth S. C., et al. 2005Analysis of Bordetella pertussis populations in European countries with different vaccination policies. J. Clin. Microbiol. 43, 2837–2843 (doi:10.1128/JCM.43.6.2837-2843.2005) [PMC free article] [PubMed]
- Van Gent M., de Greeff S. C., Van der Heide H. G., Mooi F. R. 2009An investigation into the cause of the 1983 whooping cough epidemic in the Netherlands. Vaccine 27, 1898–1903 (doi:10.1016/j.vaccine.2009.01.111) [PubMed]
- Watanabe M., Nagai M. 2004Whooping cough due to Bordetella parapertussis: an unresolved problem.Expert. Rev. Anti. Infect. Ther. 2, 447–454 (doi:10.1586/14787220.127.116.117) [PubMed]
- Webster R. G. 1966Original antigenic sin in ferrets: the response to sequential infections with influenza viruses. J. Immunol. 97, 177–183 [PubMed]
- Willems R. J., Kamerbeek J., Geuijen C. A., Top J., Gielen H., Gaastra W., Mooi F. R. 1998The efficacy of a whole cell pertussis vaccine and fimbriae against Bordetella pertussis and Bordetella parapertussisinfections in a respiratory mouse model. Vaccine 16, 410–416 (doi:10.1016/S0264-410X(97)80919-X) [PubMed]
- Wolfe D. N., Kirimanjeswara G. S., Harvill E. T. 2005Clearance of Bordetella parapertussis from the lower respiratory tract requires humoral and cellular immunity. Infect. Immun. 73, 6508–6513 (doi:10.1128/IAI.73.10.6508-6513.2005) [PMC free article] [PubMed]
- Wolfe D. N., Goebel E. M., Bjørnstad O. N., Restif O., Harvill E. T. 2007The O antigen enables Bordetella parapertussis to avoid Bordetella pertussis-induced immunity. Infect. Immun. 75, 4972–4979 (doi:10.1128/IAI.00763-07) [PMC free article] [PubMed]
- Wolfe D. N., Buboltz A. M., Harvill E. T. 2009Inefficient Toll-like receptor-4 stimulation enablesBordetella parapertussis to avoid host immunity. PLoS ONE 4, e4280 (doi:10.1371/journal.pone.0004280) [PMC free article] [PubMed]
- Zhang X., Goebel E. M., Rodriguez M. E., Preston A., Harvill E. T. 2009aThe O antigen is a critical antigen for the development of a protective immune response to Bordetella parapertussis. Infect. Immun. 77, 5050–5058 (doi:10.1128/IAI.00667-09) [PMC free article] [PubMed]
- Zhang X., Rodriguez M. E., Harvill E. T. 2009bO antigen allows B. parapertussis to evade B. pertussisvaccine-induced immunity by blocking binding and functions of cross-reactive antibodies. PLoS One 4, e6989 (doi:10.1371/journal.pone.0006989) [PMC free article] [PubMed]
- News Corp Australia has a serious conflict of interest in regards to its…
- COVID vaccine—history matters
- Censorship is shutting down concerns about vaccine safety
- A brilliant analysis of vaccination, by Richard Moskowitz, MD and homeopath
- Is it ethical to vaccinate children to protect the elderly?
- Second AstraZeneca Trial Participant Develops Rare Neurological…
- The COVID vaccine and the commercial conquest of the planet: The Plan
- “Covid-19 vaccination”an irreversible genetic modification…
About The Crazz Files
The crazz files is a totally independent Australian website & podcast show.