Bordetella bronchiseptica and Bordetella pertussis: Similarities and Differences in Infection, Immuno-Modulation, and Vaccine Considerations.

Bordetella pertussis and Bordetella bronchiseptica belong to the genus Bordetella, which comprises 14 other species. B. pertussis is responsible for whooping cough in humans, a severe infection in children and less severe or chronic in adults. These infections are restricted to humans and currently increasing worldwide. B. bronchiseptica is involved in diverse respiratory infections in a wide range of mammals. For instance, the canine infectious respiratory disease complex (CIRDC), characterized by a chronic cough in dogs. At the same time, it is increasingly implicated in human infections, while remaining an important pathogen in the veterinary field. Both Bordetella can evade and modulate host immune responses to support their persistence, although it is more pronounced in B. bronchiseptica infection. The protective immune responses elicited by both pathogens are comparable, while there are important characteristics in the mechanisms that differ. However, B. pertussis pathogenesis is more difficult to decipher in animal models than those of B. bronchiseptica because of its restriction to humans. Nevertheless, the licensed vaccines for each Bordetella are different in terms of formulation, route of administration and immune responses induced, with no known cross-reaction between them. Moreover, the target of the mucosal tissues and the induction of long-lasting cellular and humoral responses are required to control and eliminate Bordetella. In addition, the interaction between both veterinary and human fields are essential for the control of this genus, by preventing the infections in animals and the subsequent zoonotic transmission to humans.

Bystander activation of Bordetella pertussis-induced nasal tissue-resident memory CD4 T cells confers heterologous immunity to Klebsiella pneumoniae.

Tissue-resident memory CD4 T (TRM ) cells induced by infection with Bordetella pertussis persist in respiratory tissues and confer long-term protective immunity against reinfection. However, it is not clear how they are maintained in respiratory tissues. Here, we demonstrate that B. pertussis-specific CD4 TRM cells produce IL-17A in response to in vitro stimulation with LPS or heat-killed Klebsiella pneumoniae (HKKP) in the presence of dendritic cells. Furthermore, IL-17A-secreting CD4 TRM cells expand in the lung and nasal tissue of B. pertussis convalescent mice following in vivo administration of LPS or HKKP. Bystander activation of CD4 TRM cells was suppressed by anti-IL-12p40 but not by anti-MHCII antibodies. Furthermore, purified respiratory tissue-resident, but not circulating, CD4 T cells from convalescent mice produced IL-17A following direct stimulation with IL-23 and IL-1ß or IL-18. Intranasal immunization of mice with a whole-cell pertussis vaccine induced respiratory CD4 TRM cells that were reactivated following stimulation with K. pneumoniae. Furthermore, the nasal pertussis vaccine conferred protective immunity against B. pertussis but also attenuated infection with K. pneumoniae. Our findings demonstrate that CD4 TRM cells induced by respiratory infection or vaccination can undergo bystander activation and confer heterologous immunity to an unrelated respiratory pathogen.

The Mechanism of Pertussis Cough Revealed by the Mouse-Coughing Model.

Pertussis, also known as whooping cough, is a contagious respiratory disease caused by the Gram-negative bacterium Bordetella pertussis. This disease is characterized by severe and uncontrollable coughing, which imposes a significant burden on patients. However, its etiological agent and the mechanism are totally unknown because of a lack of versatile animal models that reproduce the cough. Here, we present a mouse model that reproduces coughing after intranasal inoculation with the bacterium or its components and demonstrate that lipooligosaccharide (LOS), pertussis toxin (PTx), and Vag8 of the bacterium cooperatively function to cause coughing. Bradykinin induced by LOS sensitized a transient receptor potential ion channel, TRPV1, which acts as a sensor to evoke the cough reflex. Vag8 further increased bradykinin levels by inhibiting the C1 esterase inhibitor, the major downregulator of the contact system, which generates bradykinin. PTx inhibits intrinsic negative regulation systems for TRPV1 through the inactivation of Gi GTPases. Our findings provide a basis to answer long-standing questions on the pathophysiology of pertussis cough. IMPORTANCE The Gram-negative bacterium Bordetella pertussis causes a respiratory disease called whooping cough, or pertussis. This disease is characterized by paroxysmal coughing, the mechanism of which has not been intensively studied because of a lack of versatile animal models that reproduce the cough. In this study, we present a mouse model that reproduces coughing after intranasal inoculation with the bacterium or its components. Using this model, we demonstrate that lipooligosaccharide, Vag8, and pertussis toxin of the bacteria cooperatively function to cause coughing. Our results also indicate that bradykinin, an inflammatory mediator, and TRPV1, an ion channel linked to nociceptive signaling, are host factors involved in the coughing mechanism.

Selective Enhancement of the Cell-Permeabilizing Activity of Adenylate Cyclase Toxin Does Not Increase Virulence of Bordetella pertussis.

The whooping cough agent, Bordetella pertussis, secretes an adenylate cyclase toxin-hemolysin (CyaA, ACT, or AC-Hly) that catalyzes the conversion of intracellular ATP to cAMP and through its signaling annihilates the bactericidal activities of host sentinel phagocytes. In parallel, CyaA permeabilizes host cells by the formation of cation-selective membrane pores that account for the hemolytic activity of CyaA. The pore-forming activity contributes to the overall cytotoxic effect of CyaA in vitro, and it has previously been proposed to synergize with the cAMP-elevating activity in conferring full virulence on B. pertussis in the mouse model of pneumonic infection. CyaA primarily targets myeloid phagocytes through binding of their complement receptor 3 (CR3, integrin αMß2, or CD11b/CD18). However, with a reduced efficacy, the toxin can promiscuously penetrate and permeabilize the cell membrane of a variety of non-myeloid cells that lack CR3 on the cell surface, including airway epithelial cells or erythrocytes, and detectably intoxicates them by cAMP. Here, we used CyaA variants with strongly and selectively enhanced or reduced pore-forming activity that, at the same time, exhibited a full capacity to elevate cAMP concentrations in both CR3-expressing and CR3-non-expressing target cells. Using B. pertussis mutants secreting such CyaA variants, we show that a selective enhancement of the cell-permeabilizing activity of CyaA does not increase the overall virulence and lethality of pneumonic B. pertussis infection of mice any further. In turn, a reduction of the cell-permeabilizing activity of CyaA did not reduce B. pertussis virulence any importantly. These results suggest that the phagocyte-paralyzing cAMP-elevating capacity of CyaA prevails over the cell-permeabilizing activity of CyaA that appears to play an auxiliary role in the biological activity of the CyaA toxin in the course of B. pertussis infections in vivo.

Immunogenicity of a new enhanced tetanus-reduced dose diphtheria-acellular pertussis (Tdap) vaccine against Bordetella pertussis in a murine model.

BACKGROUND: The necessity of the tetanus-reduced dose diphtheria-acellular pertussis (Tdap) vaccine in adolescence and adults has been emphasized since the resurgence of small-scale pertussis in Korea and worldwide due to the waning effect of the vaccine and variant pathogenic stains in the late 1990s. GreenCross Pharma (GC Pharma), a Korean company, developed the Tdap vaccine GC3111 in 2010. Recently, they enhanced the vaccine, GC3111, produced previously in 2010 to reinforce the antibody response against filamentous hemagglutinin (FHA). In this study, immunogenicity and efficacy of the enhanced Tdap vaccine compared and evaluated with two Tdap vaccines, GC3111 vaccine produced in 2010 previously and commercially available Tdap vaccine in a murine model. METHODS: Two tests groups and positive control group of Balb/c mice were primed with two doses of the diphtheria-tetanus-acellular pertussis (DTaP) vaccine followed by a single booster Tdap vaccine at 9 week using the commercially available Tdap vaccine or 2 Tdap vaccines from GC Pharma (GC3111, enhanced GC3111). Humoral response was assessed 1 week before and 2 and 4 weeks after Tdap booster vaccination. The enhanced GC3111 generated similar humoral response compare to the commercial vaccine for filamentous hemagglutinin (FHA). The interferon gamma (IFN-γ) (Th1), interleukin 5 (IL-5) (Th2) and interleukin 17 (IL-17) (Th17) cytokines were assessed 4 weeks after booster vaccination by stimulation with three simulators: heat inactivated Bordetella pertussis (hBp), vaccine antigens, and hBp mixed with antigens (hBp + antigen). A bacterial challenge test was performed 4 weeks after booster vaccination. RESULTS: Regarding cell-mediated immunity, cytokine secretion differed among the three simulators. However, no difference was found between two test groups and positive control group. All the vaccinated groups indicated a Th1 or Th1/Th2 response. On Day 5 post-bacterial challenge, B. pertussis colonies were absent in the lungs in two test groups and positive control group. CONCLUSIONS: Our results confirmed the immunogenicity of GC Pharma's Tdap vaccine; enhanced GC3111 was equivalent to the presently used commercial vaccine in terms of humoral response as well as cell-mediated cytokine expression.

Highly Sensitive Flow Cytometry Allows Monitoring of Changes in Circulating Immune Cells in Blood After Tdap Booster Vaccination.

Antigen-specific serum immunoglobulin (Ag-specific Ig) levels are broadly used as correlates of protection. However, in several disease and vaccination models these fail to predict immunity. In these models, in-depth knowledge of cellular processes associated with protective versus poor responses may bring added value. We applied high-throughput multicolor flow cytometry to track over-time changes in circulating immune cells in 10 individuals following pertussis booster vaccination (Tdap, Boostrix®, GlaxoSmithKline). Next, we applied correlation network analysis to extensively investigate how changes in individual cell populations correlate with each other and with Ag-specific Ig levels. We further determined the most informative cell subsets and analysis time points for future studies. Expansion and maturation of total IgG1 plasma cells, which peaked at day 7 post-vaccination, was the most prominent cellular change. Although these cells preceded the increase in Ag-specific serum Ig levels, they did not correlate with the increase of Ig levels. In contrast, strong correlation was observed between Ag-specific IgGs and maximum expansion of total IgG1 and IgA1 memory B cells at days 7 to 28. Changes in circulating T cells were limited, implying the need for a more sensitive approach. Early changes in innate immune cells, i.e. expansion of neutrophils, and expansion and maturation of monocytes up to day 5, most likely reflected their responses to local damage and adjuvant. Here we show that simultaneous monitoring of multiple circulating immune subsets in blood by flow cytometry is feasible. B cells seem to be the best candidates for vaccine monitoring.

Reinvestigating the Coughing Rat Model of Pertussis To Understand Bordetella pertussis Pathogenesis.

Bordetella pertussis is a highly contagious bacterium that is the causative agent of whooping cough (pertussis). Currently, acellular pertussis vaccines (aP, DTaP, and Tdap) are used to prevent pertussis disease. However, it is clear that the aP vaccine efficacy quickly wanes, resulting in the reemergence of pertussis. Furthermore, recent work performed by the CDC suggest that current circulating strains are genetically distinct from strains of the past. The emergence of genetically diverging strains, combined with waning aP vaccine efficacy, calls for reevaluation of current animal models of pertussis. In this study, we used the rat model of pertussis to compare two genetically divergent strains Tohama 1 and D420. We intranasally challenged 7-week-old Sprague-Dawley rats with 108 viable Tohama 1 and D420 and measured the hallmark signs/symptoms of B. pertussis infection such as neutrophilia, pulmonary inflammation, and paroxysmal cough using whole-body plethysmography. Onset of cough occurred between 2 and 4 days after B. pertussis challenge, averaging five coughs per 15 min, with peak coughing occurring at day 8 postinfection, averaging upward of 13 coughs per 15 min. However, we observed an increase of coughs in rats infected with clinical isolate D420 through 12 days postchallenge. The rats exhibited increased bronchial restriction following B. pertussis infection. Histology of the lung and flow cytometry confirm both cellular infiltration and pulmonary inflammation. D420 infection induced higher production of anti-B. pertussis IgM antibodies compared to Tohama 1 infection. The coughing rat model provides a way of characterizing disease manifestation differences between B. pertussis strains.

Circulation of pertussis and poor protection against diphtheria among middle-aged adults in 18 European countries.

Reported incidence of pertussis in the European Union (EU) and the European Economic Area (EEA) varies and may not reflect the real situation, while vaccine-induced protection against diphtheria and tetanus seems sufficient. We aimed to determine the seroprevalence of DTP antibodies in EU/EEA countries within the age groups of 40-49 and 50-59 years. Eighteen countries collected around 500 samples between 2015 and 2018 (N = 10,302) which were analysed for IgG-DTP specific antibodies. The proportion of sera with pertussis toxin antibody levels ≥100 IU/mL, indicative of recent exposure to pertussis was comparable for 13/18 countries, ranging between 2.7-5.8%. For diphtheria the proportion of sera lacking the protective level (<0.1 IU/mL) varied between 22.8-82.0%. For tetanus the protection was sufficient. Here, we report that the seroprevalence of pertussis in these age groups indicates circulation of B. pertussis across EU/EEA while the lack of vaccine-induced seroprotection against diphtheria is of concern and deserves further attention.

Omics Analysis of Blood-Responsive Regulon in Bordetella pertussis Identifies a Novel Essential T3SS Substrate.

Bacterial pathogens sense specific cues associated with different host niches and integrate these signals to appropriately adjust the global gene expression. Bordetella pertussis is a Gram-negative, strictly human pathogen of the respiratory tract and the etiological agent of whooping cough (pertussis). Though B. pertussis does not cause invasive infections, previous results indicated that this reemerging pathogen responds to blood exposure. Here, omics RNA-seq and LC-MS/MS techniques were applied to determine the blood-responsive regulon of B. pertussis. These analyses revealed that direct contact with blood rewired global gene expression profiles in B. pertussis as the expression of almost 20% of all genes was significantly modulated. However, upon loss of contact with blood, the majority of blood-specific effects vanished, with the exception of several genes encoding the T3SS-secreted substrates. For the first time, the T3SS regulator BtrA was identified in culture supernatants of B. pertussis. Furthermore, proteomic analysis identified BP2259 protein as a novel secreted T3SS substrate, which is required for T3SS functionality. Collectively, presented data indicate that contact with blood represents an important cue for B. pertussis cells.

Lack of evidence supporting a role of IFN-ß and TGF-ß in differential polarization of Bordetella pertussis specific-T cell responses.

Bordetella Pertussis (BP) vaccine-induced immunity is waning worldwide despite excellent vaccine coverage. Replacement of the whole-cell inactivated vaccine (wP) by an acellular subunit vaccine (aP) is thought to play a major role and to be associated with the recurrence of whooping cough. Previously, we detected that the polarization towards a Th2 and Th1/Th17 response in aP and wP vaccinees, respectively, persists upon aP boosting in adolescents and adults. Additionally, IL-9 and TGF-ß were found to be up-regulated in aP-primed donors and network analysis further identified IFN-ß as a potential upstream regulator of IL-17 and IL-9. Based on these findings, we hypothesized that IFN-ß produced following aP vaccination may lead to increased IL-9 and decreased IL-17 production. Also, due to the well characterized role of TGF-ß in both Th17 and Th9 differentiation, we put forth that TGF-ß addition to BP-stimulated CD4 + T cells might modulate IL-17 and IL-9 production. To test this hypothesis, we stimulated in vitro cultures of PBMC or isolated naive CD4 + T cells from aP vs wP donors with a pool of BP epitopes and assessed the effect of IFN-ß or TGF-ß in proliferative responses as well as in the cytokine secretion of IL-4, IL-9, IL-17, and IFN-γ. IFN-ß reduced BP-specific proliferation in PBMC as well as cytokine production but increased IL-9, IL-4, and IFN-γ cytokines in naïve CD4 + T cells. These effects were independent of the childhood vaccination received by the donors. Similarly, TGF-ß reduced BP-specific proliferation in PBMC but induced proliferation in naïve CD4 + T cells. However, stimulation was associated with a generalized inhibition of cytokine production regardless of the original aP or wP vaccination received by the donors. Our study suggests that key T cell functions such as cytokine secretion are under the control of antigen stimulation and environmental cues but molecular pathways different than the ones investigated here might underlie the long-lasting differential cytokine production associated with aP- vs wP-priming in childhood vaccination.

Risk factors for Bordetella pertussis disease in hospitalized children.

INTRODUCTION: Despite a resurgence of disease, risk factors for pertussis in children in low and middle-income countries are poorly understood. This study aimed to investigate risk factors for pertussis disease in African children hospitalized with severe LRTI. METHODS: A prospective study of children hospitalized with severe LRTI in Cape Town, South Africa was conducted over a one-year period. Nasopharyngeal and induced sputum samples from child and nasopharyngeal sample from caregiver were tested for Bordetella pertussis using PCR (IS481+/hIS1001). History and clinical details were documented. RESULTS: 460 children with a median age of 8 (IQR 4-18) months were enrolled. B. pertussis infection was confirmed in 32 (7.0%). The adjusted risk of confirmed pertussis was significantly increased if infants were younger than two months [aRR 2.37 (95% CI 1.03-5.42]), HIV exposed but uninfected (aRR 3.53 [95% CI 1.04-12.01]) or HIV infected (aRR 4.35 [95% CI 1.24-15.29]). Mild (aRR 2.27 [95% CI 1.01-5.09]) or moderate (aRR 2.70 [95% CI 1.13-6.45]) under-nutrition in the children were also associated with higher risk. The highest adjusted risk occurred in children whose caregivers had B. pertussis detected from nasopharyngeal swabs (aRR 13.82 [95% CI 7.76-24.62]). Completion of the primary vaccine schedule (three or more doses) was protective (aRR 0.28 [95% CI 0.10-0.75]). CONCLUSIONS: HIV exposure or infection, undernutrition as well as detection of maternal nasal B. pertussis were associated with increased risk of pertussis in African children, especially in young infants. Completed primary vaccination was protective. There is an urgent need to improve primary pertussis vaccine coverage in low and middle-income countries. Pertussis vaccination of pregnant women, especially those with HIV infection should be prioritized.

Adenylate Cyclase Toxin Tinkering With Monocyte-Macrophage Differentiation.

Circulating inflammatory monocytes are attracted to infected mucosa and differentiate into macrophage or dendritic cells endowed with enhanced bactericidal and antigen presenting capacities. In this brief Perspective we discuss the newly emerging insight into how the cAMP signaling capacity of Bordetella pertussis adenylate cyclase toxin manipulates the differentiation of monocytes and trigger dedifferentiation of the alveolar macrophages to facilitate bacterial colonization of human airways.

Effect of FHA and Prn on Bordetella pertussis colonization of mice is dependent on vaccine type and anatomical site.

Bordetella pertussis vaccine escape mutants that lack expression of the pertussis antigen pertactin (Prn) have emerged in vaccinated populations in the last 10-20 years. Additionally, clinical isolates lacking another acellular pertussis (aP) vaccine component, filamentous hemagglutinin (FHA), have been found sporadically. Here, we show that both whole-cell pertussis (wP) and aP vaccines induced protection in the lungs of mice, but that the wP vaccine was more effective in nasal clearance. Importantly, bacterial populations isolated from the lungs shifted to an FHA-negative phenotype due to frameshift mutations in the fhaB gene. Loss of FHA expression was strongly selected for in Prn-deficient strains in the lungs following aP but not wP vaccination. The combined loss of Prn and FHA led to complete abrogation of bacterial surface binding by aP-induced serum antibodies. This study demonstrates vaccine- and anatomical site-dependent adaptation of B. pertussis and has major implications for the design of improved pertussis vaccines.

Cytotoxicity of the effector protein BteA was attenuated in Bordetella pertussis by insertion of an alanine residue.

Bordetella bronchiseptica and Bordetella pertussis are closely related respiratory pathogens that evolved from a common bacterial ancestor. While B. bronchiseptica has an environmental reservoir and mostly establishes chronic infections in a broad range of mammals, B. pertussis is a human-specific pathogen causing acute pulmonary pertussis in infants and whooping cough illness in older humans. Both species employ a type III secretion system (T3SS) to inject a cytotoxic BteA effector protein into host cells. However, compared to the high BteA-mediated cytotoxicity of B. bronchiseptica, the cytotoxicity induced by B. pertussis BteA (Bp BteA) appears to be quite low and this has been attributed to the reduced T3SS gene expression in B. pertussis. We show that the presence of an alanine residue inserted at position 503 (A503) of Bp BteA accounts for its strongly attenuated cytotoxic potency. The deletion of A503 from Bp BteA greatly enhanced the cytotoxic activity of B. pertussis B1917 on mammalian HeLa cells and expression of Bp BteAΔA503 was highly toxic to Saccharomyces cerevisiae cells. Vice versa, insertion of A503 into B. bronchiseptica BteA (Bb BteA) strongly decreased its cytotoxicity to yeast and HeLa cells. Moreover, the production of Bp BteAΔA503 increased virulence of B. pertussis B1917 in the mouse model of intranasal infection (reduced LD50) but yielded less inflammatory pathology in infected mouse lungs at sublethal infectious doses. This suggests that A503 insertion in the T3SS effector Bp BteA may represent an evolutionary adaptation that fine-tunes B. pertussis virulence and host immune response.

Transcriptional profiling of human macrophages during infection with Bordetella pertussis.

Bordetella pertussis, a strictly human re-emerging pathogen and the causative agent of whooping cough, exploits a broad variety of virulence factors to establish efficient infection. Here, we used RNA sequencing to analyse the changes in gene expression profiles of human THP-1 macrophages resulting from B. pertussis infection. In parallel, we attempted to determine the changes in intracellular B. pertussis-specific transcriptomic profiles resulting from interaction with macrophages. Our analysis revealed that global gene expression profiles in THP-1 macrophages are extensively rewired 6 h post-infection. Among the highly expressed genes, we identified those encoding cytokines, chemokines, and transcription regulators involved in the induction of the M1 and M2 macrophage polarization programmes. Notably, several host genes involved in the control of apoptosis and inflammation which are known to be hijacked by intracellular bacterial pathogens were overexpressed upon infection. Furthermore, in silico analyses identified large temporal changes in expression of specific gene subsets involved in signalling and metabolic pathways. Despite limited numbers of the bacterial reads, we observed reduced expression of majority of virulence factors and upregulation of several transcriptional regulators during infection suggesting that intracellular B. pertussis cells switch from virulent to avirulent phase and actively adapt to intracellular environment, respectively.

T Cell Metabolism Is Dependent on Anatomical Location within the Lung.

The metabolic shift from oxidative phosphorylation to glycolysis is universally accepted as a necessary step for immune cells to mount effector functions. However, it is unknown if this paradigm holds true for T cells regardless of anatomical location. In this study, we compared metabolic responses among distinct mouse pulmonary CD4+ effector T cell (Teff) pools following intranasal vaccination with either Francisella tularensis or Bordetella pertussis Surprisingly, in contrast to circulating CD4+ Teff, upon ex vivo stimulation, resident CD4+ Teff did not shift to glycolysis. This impairment in the resident pool was modestly overcome following in vivo infection. However, consistent with an ex vivo triggered shift toward glycolysis, circulating CD4+ Teff remained superior compared with resident CD4+ Teff after in vivo infection. These data indicate differences in lung T cell metabolism is associated with anatomic location, a feature which may be exploited to enhance or dampen pulmonary T cell responses.

Bordetella pertussis Can Be Motile and Express Flagellum-Like Structures.

Bordetella bronchiseptica encodes and expresses a flagellar apparatus. In contrast, Bordetella pertussis, the causative agent of whooping cough, has historically been described as a nonmotile and nonflagellated organism. The previous statements that B. pertussis was a nonmotile organism were consistent with a stop codon located in the flagellar biosynthesis gene, flhA, discovered when the B. pertussis Tohama I genome was sequenced and analyzed by Parkhill et al. in 2003 (J. Parkhill, M. Sebaihia, A. Preston, L. D. Murphy, et al., Nat Genet, 35:32-40, 2003, https://doi.org/10.1038/ng1227). The stop codon has subsequently been found in all annotated genomes. Parkhill et al. also showed, however, that B. pertussis contains all genetic material required for flagellar synthesis and function. We and others have determined by various transcriptomic analyses that these flagellar genes are differentially regulated under a variety of B. pertussis growth conditions. In light of these data, we tested for B. pertussis motility and found that both laboratory-adapted strains and clinical isolates can be motile. Upon isolation of motile B. pertussis, we discovered flagellum-like structures on the surface of the bacteria. B. pertussis motility appears to occur primarily in the Bvg(-) phase, consistent with regulation present in B. bronchiseptica Motility can also be induced by the presence of fetal bovine serum. These observations demonstrate that B. pertussis can express flagellum-like structures, and although it remains to be determined if B. pertussis expresses flagella during infection or if motility and/or flagella play roles during the cycle of infection and transmission, it is clear that these data warrant further investigation.IMPORTANCE This report provides evidence for motility and expression of flagella by B. pertussis, a bacterium that has been reported as nonmotile since it was first isolated and studied. As with B. bronchiseptica, B. pertussis cells can express and assemble a flagellum-like structure on their surface, which in other organisms has been implicated in several important processes that occur in vivo The discovery that B. pertussis is motile raises many questions, including those regarding the mechanisms of regulation for flagellar gene and protein expression and, importantly, the role of flagella during infection. This novel observation provides a foundation for further study of Bordetella flagella and motility in the contexts of infection and transmission.

Pertussis vaccines and protective immunity.

Despite high vaccine coverage, reported cases of pertussis have increased steadily over the last twenty years. This resurgence has stimulated interest in host responses to pertussis infection and vaccination with the goal of developing more effective next-generation vaccines and vaccination strategies. Optimal protection against Bordetella pertussis appears to be multifactorial requiring both humoral and cellular responses. Natural infection and whole-cell pertussis vaccination induce Th1 and Th17-dominated responses. In contrast, acellular vaccines induce Th2-dominated responses. Available immunological data indicate that while antibodies provide protection against disease, Th1 and Th17-mediated immune responses are required for bacterial clearance and long-lasting protection. The nature of the priming in children appears to be important in modulating bias and durability of immune responses required to provide protection against B. pertussis. This review summarizes the current understanding of differences in immune responses and their role in protection against B. pertussis following infection or vaccination.

Evaluation of Pertussis Toxin Expression in B2 and THIJS Media.

Whole-cell pertussis vaccine (wP) has been imperative and highly effective in preventing childhood deaths due to pertussis. Pertussis toxin is one of the virulence factors of Bordetella pertussis in all available pertussis vaccines. wP production in Razi Vaccine and Serum Research Institute is according to bioreactor culture of B. pertussis strains in B2 medium. The aim of this study was to evaluate B. pertussis strain 509 PT production in B2 and Thalen-IJssel (THIJS) media by Chinese Hamster Ovary (CHO) cell and enzyme-linked immunosorbent assay methods (ELISA). In the current study, B. pertussis strain 509 was cultured in B2 and THIJS media. Six samples were taken during the log growth phase within 2-3 h intervals (triplicate). The growth rate was calculated using opacity and the quantification of cell-associated and released PT measured by ELISA and CHO cell assays. THIJS medium was significantly showed an increase in the bacterial growth rate. During the first 29 h, bacterial concentrations in B2 and THIJS culture medium were 19 and 29 IOU, respectively. In THIJS medium, greater amount of pertussistoxin production was cell-associated. In B2 medium, maximum cell-associated toxin by ELISA and CHO cell assays were in the ODs of 1.1 and 0.9 and for THIJS medium in the ODs of 1.6 and 1.1, respectively. B. pertussis strain 509 in THIJS medium produced higher cell mass and cell-associated pertussis toxin than that of B2. It can be used for the production of whole-cell vaccine with higher pertussis toxin and accordingly using lower biomass per dose leading to the reduction of vaccine toxicity.

Genotypic and phenotypic adaptation of pathogens: lesson from the genus Bordetella.

PURPOSE OF REVIEW: To relate genomic changes to phenotypic adaptation and evolution from environmental bacteria to obligate human pathogens, focusing on the examples within Bordetella species. RECENT FINDINGS: Recent studies showed that animal-pathogenic and human-pathogenic Bordetella species evolved from environmental ancestors in soil. The animal-pathogenic Bordetella bronchiseptica can hijack the life cycle of the soil-living amoeba Dictyostelium discoideum, surviving inside single-celled trophozoites, translocating to the fruiting bodies and disseminating along with amoeba spores. The association with amoeba may have been a 'training ground' for bacteria during the evolution to pathogens. Adaptation to an animal-associated life style was characterized by decreasing metabolic versatility and genome size and by acquisition of 'virulence factors' mediating the interaction with the new animal hosts. Subsequent emergence of human-specific pathogens, such as Bordetella pertussis from zoonoses of broader host range progenitors, was accompanied by a dramatic reduction in genome size, marked by the loss of hundreds of genes. SUMMARY: The evolution of Bordetella from environmental microbes to animal-adapted and obligate human pathogens was accompanied by significant genome reduction with large-scale gene loss during divergence.