The contribution of BvgR, RisA, and RisS to global gene regulation, intracellular cyclic-di-GMP levels, motility, and biofilm formation in Bordetella bronchiseptica.

Bordetella bronchiseptica is a highly contagious respiratory bacterial veterinary pathogen. In this study the contribution of the transcriptional regulators BvgR, RisA, RisS, and the phosphorylation of RisA to global gene regulation, intracellular cyclic-di-GMP levels, motility, and biofilm formation were evaluated. Next Generation Sequencing (RNASeq) was used to differentiate the global gene regulation of both virulence-activated and virulence-repressed genes by each of these factors. The BvgAS system, along with BvgR, RisA, and the phosphorylation of RisA served in cyclic-di-GMP degradation. BvgR and unphosphorylated RisA were found to temporally regulate motility. Additionally, BvgR, RisA, and RisS were found to be required for biofilm formation.

Repeated Bordetella pertussis Infections Are Required to Reprogram Acellular Pertussis Vaccine-Primed Host Responses in the Baboon Model.

BACKGROUND: The United States has experienced a resurgence of pertussis following the introduction of acellular pertussis (aP) vaccines. This is likely due to the failure of aP vaccines to induce durable immunity and prevent infection, carriage, and transmission. METHODS: To evaluate the impact of aP vaccination on the immune response to infection and test the ability of infection to reprogram aP-imprinted immune responses, we challenged unvaccinated and aP-vaccinated baboons with Bordetella pertussis multiple times and accessed the immune responses and outcomes of infections after each exposure. RESULTS: Multiple infections were required to elicit T-helper 17 responses and protection in aP-vaccinated animals comparable to responses seen in unvaccinated animals after a single challenge. Even after 3 challenges, T-helper 1 responses were not observed in aP-vaccinated animals. Immunoglobulin G responses to vaccine and nonvaccine antigens were not negatively affected in aP-vaccinated animals. CONCLUSIONS: Our results indicate that it is possible to retrain aP-primed immune responses, but it will likely require an optimal booster and multiple doses. Our results in the baboon model suggest that circulation of B. pertussis in aP-vaccinated populations is concentrated in the younger age bands of the population, providing information that can guide improved modeling of B. pertussis epidemiology in aP-vaccinated populations.

T cell reactivity to Bordetella pertussis is highly diverse regardless of childhood vaccination.

The incidence of whooping cough due to Bordetella pertussis (BP) infections has increased recently. It is believed that the shift from whole-cell pertussis (wP) vaccines to acellular pertussis (aP) vaccines may be contributing to this rise. While T cells are key in controlling and preventing disease, nearly all knowledge relates to antigens in aP vaccines. A whole-genome mapping of human BP-specific CD4+ T cell responses was performed in healthy vaccinated adults and revealed unexpected broad reactivity to hundreds of antigens. The overall pattern and magnitude of T cell responses to aP and non-aP vaccine antigens are similar regardless of childhood vaccination, suggesting that asymptomatic infections drive the pattern of T cell reactivity in adults. Lastly, lack of Th1/Th2 polarization to non-aP vaccine antigens suggests these antigens have the potential to counteract aP vaccination Th2 bias. These findings enhance our insights into human T cell responses to BP and identify potential targets for next-generation pertussis vaccines.

Antigen Discovery for Next-Generation Pertussis Vaccines Using Immunoproteomics and Transposon-Directed Insertion Sequencing.

BACKGROUND: Despite high vaccination rates, the United States has experienced a resurgence in reported cases of pertussis after switching to the acellular pertussis vaccine, indicating a need for improved vaccines that enhance infection control. METHODS: Bordetella pertussis antigens recognized by convalescent-baboon serum and nasopharyngeal wash were identified by immunoproteomics and their subcellular localization predicted. Genes essential or important for persistence in the baboon airway were identified by transposon-directed insertion-site sequencing (TraDIS) analysis. RESULTS: In total, 314 B. pertussis antigens were identified by convalescent baboon serum and 748 by nasopharyngeal wash. Thirteen antigens were identified as immunogenic in baboons, essential for persistence in the airway by TraDIS, and membrane-localized: BP0840 (OmpP), Pal, OmpA2, BP1485, BamA, Pcp, MlaA, YfgL, BP2197, BP1569, MlaD, ComL, and BP0183. CONCLUSIONS: The B. pertussis antigens identified as immunogenic, essential for persistence in the airway, and membrane-localized warrant further investigation for inclusion in vaccines designed to reduce or prevent carriage of bacteria in the airway of vaccinated individuals.

Pertussis Toxin: A Key Component in Pertussis Vaccines?

B. pertussis is a human-specific pathogen and the causative agent of whooping cough. The ongoing resurgence in pertussis incidence in high income countries is likely due to faster waning of immunity and increased asymptomatic colonization in individuals vaccinated with acellular pertussis (aP) vaccine relative whole-cell pertussis (wP)-vaccinated individuals. This has renewed interest in developing more effective vaccines and treatments and, in support of these efforts, defining pertussis vaccine correlates of protection and the role of vaccine antigens and toxins in disease. Pertussis and its toxins have been investigated by scientists for over a century, yet we still do not have a clear understanding of how pertussis toxin (PT) contributes to disease symptomology or how anti-PT immune responses confer protection. This review covers PT's role in disease and evidence for its protective role in vaccines. Clinical data suggest that PT is a defining and essential toxin for B. pertussis pathogenesis and, when formulated into a vaccine, can prevent disease. Additional studies are required to further elucidate the role of PT in disease and vaccine-mediated protection, to inform the development of more effective treatments and vaccines.

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.

Histopathology of Bordetella pertussis in the Baboon Model.

Pertussis is a severe respiratory disease caused by Bordetella pertussis The classic symptoms of pertussis include paroxysmal coughing with an inspiratory whoop, posttussive vomiting, cyanosis, and persistent coryzal symptoms. Infants under 2 months of age experience more severe disease, with most deaths occurring in this age group. Most of what is known about the pathology of pertussis in humans is from the evaluation of fatal human infant cases. The baboon model of pertussis provides the opportunity to evaluate the histopathology of severe but nonfatal pertussis. The baboon model recapitulates the characteristic clinical signs of pertussis observed in humans, including leukocytosis, paroxysmal coughing, mucus production, heavy colonization of the airway, and transmission of the bacteria between hosts. As in humans, baboons demonstrate age-related differences in clinical presentation, with younger animals experiencing more severe disease. We examined the histopathology of 5- to 6-week-old baboons, with the findings being similar to those reported for fatal human infant cases. In juvenile baboons, we found that the disease is highly inflammatory and concentrated to the lungs with signs of disease that would typically be diagnosed as acute respiratory distress syndrome (ARDS) and bronchopneumonia. In contrast, no significant pathology was observed in the trachea. Histopathological changes in the trachea were limited to cellular infiltrates and mucus production. Immunohistostaining revealed that the bacteria were localized to the surface of the ciliated epithelium in the conducting airways. Our observations provide important insights into the pathology of pertussis in typical, severe but nonfatal pertussis cases in a very relevant animal model.

Maternal Vaccination With a Monocomponent Pertussis Toxoid Vaccine Is Sufficient to Protect Infants in a Baboon Model of Whooping Cough.

Background: Bordetella pertussis is a human pathogen responsible for serious respiratory illness. The disease is most severe in infants too young to be vaccinated with most hospitalizations and deaths occurring within this age group. The Advisory Committee on Immunization Practices recommended immunization of pregnant women to protect infants from birth until their first vaccination at 6-8 weeks of age. We previously demonstrated that maternal vaccination with licensed acellular pertussis vaccines protected newborn baboons from disease. We hypothesized that protection was due to toxin-neutralizing, maternal anti-pertussis toxin antibodies and predicted that maternal vaccination with a pertussis toxoid (PTx)-only vaccine would protect newborns from disease. Methods: Infant baboons born to unvaccinated mothers or mothers vaccinated with a PTx-only vaccine were challenged with B. pertussis at 5 weeks of age and followed for infection and signs of disease. Results: Although all challenged infants were heavily colonized, the infant baboons born to mothers vaccinated with PTx-only vaccine were free from clinical disease following exposure to B. pertussis. In contrast, disease was observed in infants born to unvaccinated mothers. Conclusions: Our results demonstrated that maternal vaccination with a PTx-only vaccine is sufficient to protect newborn baboons from disease following exposure to pertussis.

Activation of Bvg-Repressed Genes in Bordetella pertussis by RisA Requires Cross Talk from Noncooperonic Histidine Kinase RisK.

The two-component response regulator RisA, encoded by open reading frame BP3554 in the Bordetella pertussis Tohama I genomic sequence, is a known activator of vrg genes, a set of genes whose expression is increased under the same environmental conditions (known as modulation) that result in repression of the bvgAS virulence regulon. Here we demonstrate that RisA is phosphorylated in vivo and that RisA phosphorylation is required for activation of vrg genes. An adjacent histidine kinase gene, risS, is truncated by frameshift mutation in B. pertussis but not in Bordetella bronchiseptica or Bordetella parapertussis Neither deletion of risS' or bvgAS nor phenotypic modulation with MgSO4 affected levels of phosphorylated RisA (RisA∼P) in B. pertussis However, RisA phosphorylation did require the histidine kinase encoded by BP3223, here named RisK (cognate histidine kinase of RisA). RisK was also required for expression of the vrg genes. This requirement could be obviated by the introduction of the phosphorylation-mimicking RisAD60E mutant, indicating that an active conformation of RisA, but not phosphorylation per se, is crucial for vrg activation. Interestingly, expression of vrg genes is still modulated by MgSO4 in cells harboring the RisAD60E mutation, suggesting that the activated RisA senses additional signals to control vrg expression in response to environmental stimuli.IMPORTANCE In B. pertussis, the BvgAS two-component system activates the expression of virulence genes by binding of BvgA∼P to their promoters. Expression of the reciprocally regulated vrg genes requires RisA and is also repressed by the Bvg-activated BvgR. RisA is an OmpR-like response regulator, but RisA phosphorylation was not expected because the gene for its presumed, cooperonic, histidine kinase is inactivated by mutation. In this study, we demonstrate phosphorylation of RisA in vivo by a noncooperonic histidine kinase. We also show that RisA phosphorylation is necessary but not sufficient for vrg activation but, importantly, is not affected by BvgAS status. Instead, we propose that vrg expression is controlled by BvgAS through its regulation of BvgR, a cyclic di-GMP (c-di-GMP) phosphodiesterase.

Pertussis disease and transmission and host responses: insights from the baboon model of pertussis.

Whooping cough is a highly contagious, acute respiratory disease, caused by the Gram-negative bacterium Bordetella pertussis (Bp). Despite the introduction and widespread use of vaccines starting in the 1950s pertussis cases continue to be reported, with a significant global impact. The role of specific virulence factors in disease and the immune mechanisms associated with protection following natural infection or vaccination are still not completely understood. The recently-developed baboon model of clinical pertussis provides a valuable tool for the study of pertussis. Baboons infected with B. pertussis exhibit all of the manifestations of human pertussis including paroxysmal coughing, mucus production, leukocytosis and transmission. The establishment of this model provides the opportunity to address unanswered questions about the natural progression of this disease and host responses to infection and vaccination in a very relevant model. In this review, we present an overview of our knowledge of pertussis along with recent advances resulting from use of the baboon model. Remaining questions and future research directions are discussed. We hope that the knowledge gained through use of the baboon model of pertussis and clinical studies will allow the development of more efficacious vaccines, conferring long lasting protection against disease and transmission.

Use of a Toxin Neutralization Assay To Characterize the Serologic Response to Adenylate Cyclase Toxin after Infection with Bordetella pertussis.

Adenylate cyclase toxin (ACT) is an essential virulence factor of Bordetella pertussis, and antibodies to ACT protect against B. pertussis infection in mice. The toxin is therefore a strong candidate antigen for addition to future acellular pertussis vaccines. In order to characterize the functionality of the immunologic response to ACT after infection, we developed an assay for testing the ability of serum samples from subjects infected with B. pertussis to neutralize ACT-induced cytotoxicity in J774 macrophage cells. Baboons develop neutralizing anti-ACT antibodies following infection with B. pertussis, and all sera from baboons with positive anti-ACT IgG enzyme-linked immunosorbent assay (ELISA) results neutralized ACT cytotoxicity. The toxin neutralization assay (TNA) was positive in some baboon sera in which ELISA remained negative. Of serum samples obtained from humans diagnosed with pertussis by PCR, anti-ACT IgG ELISA was positive in 72%, and TNA was positive in 83%. All samples positive for anti-ACT IgG ELISA were positive by TNA, and none of the samples from humans without pertussis neutralized toxin activity. These findings indicate that antibodies to ACT generated following infection with B. pertussis consistently neutralize toxin-induced cytotoxicity and that TNA can be used to improve understanding of the immunologic response to ACT after infection or vaccination.

Highlights of the 11th International Bordetella Symposium: from Basic Biology to Vaccine Development.

Pertussis is a severe respiratory disease caused by infection with the bacterial pathogen Bordetella pertussis The disease affects individuals of all ages but is particularly severe and sometimes fatal in unvaccinated young infants. Other Bordetella species cause diseases in humans, animals, and birds. Scientific, clinical, public health, vaccine company, and regulatory agency experts on these pathogens and diseases gathered in Buenos Aires, Argentina from 5 to 8 April 2016 for the 11th International Bordetella Symposium to discuss recent advances in our understanding of the biology of these organisms, the diseases they cause, and the development of new vaccines and other strategies to prevent these diseases. Highlights of the meeting included pertussis epidemiology in developing nations, genomic analysis of Bordetella biology and evolution, regulation of virulence factor expression, new model systems to study Bordetella biology and disease, effects of different vaccines on immune responses, maternal immunization as a strategy to prevent newborn disease, and novel vaccine development for pertussis. In addition, the group approved the formation of an International Bordetella Society to promote research and information exchange on bordetellae and to organize future meetings. A new Bordetella.org website will also be developed to facilitate these goals.

The multifaceted RisA regulon of Bordetella pertussis.

The whooping cough agent Bordetella pertussis regulates the production of its virulence factors by the BvgA/S system. Phosphorylated BvgA activates the virulence-activated genes (vags) and represses the expression of the virulence-repressed genes (vrgs) via the activation of the bvgR gene. In modulating conditions, with MgSO4, the BvgA/S system is inactive, and the vrgs are expressed. Here, we show that the expression of almost all vrgs depends on RisA, another transcriptional regulator. We also show that some vags are surprisingly no longer modulated by MgSO4 in the risA(-) background. RisA also regulates the expression of other genes, including chemotaxis and flagellar operons, iron-regulated genes, and genes of unknown function, which may or may not be controlled by BvgA/S. We identified RisK as the likely cognate RisA kinase and found that it is important for expression of most, but not all RisA-regulated genes. This was confirmed using the phosphoablative RisAD(60)N and the phosphomimetic RisAD(60)E analogues. Thus the RisA regulon adds a new layer of complexity to B. pertussis virulence gene regulation.

Comparison of Three Whole-Cell Pertussis Vaccines in the Baboon Model of Pertussis.

Pertussis is a highly contagious respiratory illness caused by the bacterial pathogen Bordetella pertussis. Pertussis rates in the United States have escalated since the 1990s and reached a 50-year high of 48,000 cases in 2012. While this pertussis resurgence is not completely understood, we previously showed that the current acellular pertussis vaccines do not prevent colonization or transmission following challenge. In contrast, a whole-cell pertussis vaccine accelerated the rate of clearance compared to rates in unvaccinated animals and animals treated with the acellular vaccine. In order to understand if these results are generalizable, we used our baboon model to compare immunity from whole-cell vaccines from three different manufacturers that are approved outside the United States. We found that, compared to clearance rates with no vaccine and with an acellular pertussis vaccine, immunization with any of the three whole-cell vaccines significantly accelerated the clearance of B. pertussis following challenge. Whole-cell vaccination also significantly reduced the total nasopharyngeal B. pertussis burden, suggesting that these vaccines reduce the opportunity for pertussis transmission. Meanwhile, there was no difference in either the duration or in B. pertussis burden between unvaccinated and acellular-pertussis-vaccinated animals, while previously infected animals were not colonized following reinfection. We also determined that transcription of the gene encoding interleukin-17 (IL-17) was increased in whole-cell-vaccinated and previously infected animals but not in acellular-pertussis-vaccinated animals following challenge. Together with our previous findings, these data are consistent with a role for Th17 responses in the clearance of B. pertussis infection.

Bordetella pertussis transmission.

Bordetella pertussis and B. bronchiseptica are Gram-negative bacterial respiratory pathogens. Bordetella pertussis is the causative agent of whooping cough and is considered a human-adapted variant of B. bronchiseptica. Bordetella pertussis and B. bronchiseptica share mechanisms of pathogenesis and are genetically closely related. However, despite the close genetic relatedness, these Bordetella species differ in several classic fundamental aspects of bacterial pathogens such as host range, pathologies and persistence. The development of the baboon model for the study of B. pertussis transmission, along with the development of the swine and mouse model for the study of B. bronchiseptica, has enabled the investigation of different aspects of transmission including the route, attack rate, role of bacterial and host factors, and the impact of vaccination on transmission. This review will focus on B. pertussis transmission and how animal models of B. pertussis transmission and transmission models using the closely related B. bronchiseptica have increased our understanding of B. pertussis transmission.

Complete Genome Sequence of Bordetella pertussis D420.

Bordetella pertussis is the causative agent of whooping cough, a highly contagious, acute respiratory illness that has seen resurgence despite the use of vaccines. We present the complete genome sequence of a clinical strain of B. pertussis, D420, which is representative of a currently circulating clade of this pathogen.

The baboon model of pertussis: effective use and lessons for pertussis vaccines.

The USA is experiencing a pertussis resurgence that resulted in a 60-year high of 48,000 cases in 2012. Our ability to counteract this resurgence is hampered by the fact that pertussis pathogenesis and immunity to pertussis infection are not well studied. Studies in humans are difficult due to the low frequency of pertussis in the population, the cyclical nature of incidence and the sporadic geographic distribution of cases. While existing animal models reproduce many aspects of pertussis, none of them adequately reproduces the full spectrum of disease. We describe the baboon model of pertussis. The baboon model is the first animal model that recapitulates the full spectrum of human pertussis including coughing and transmission. This model is being utilized to examine pertussis pathogenesis and host responses to infection and vaccination. It is likely the baboon model will provide an important tool in the development of improved pertussis vaccines.