Alpha-1 antitrypsin inhibits pertussis toxin.

Pertussis (whooping cough) is a vaccine-preventable but re-emerging, highly infectious respiratory disease caused by Bordetella pertussis. There are currently no effective treatments for pertussis, complicating care for non-vaccinated individuals, especially newborns. Disease manifestations are predominantly caused by pertussis toxin (PT), a pivotal virulence factor classified as an ADP-ribosylating AB-type protein toxin. In this work, an unbiased approach using peptide libraries, bioassay-guided fractionation and mass spectrometry revealed α1-antitrypsin (α1AT) as a potent PT inhibitor. Biochemistry-, cell culture- and molecular modeling-based in vitro experimentation demonstrated that the α1AT mode of action is based on blocking PT-binding to the host target cell surface. In the infant mouse model of severe pertussis, α1AT expression was reduced upon infection. Further, systemic administration of α1AT significantly reduced B. pertussis-induced leukocytosis, which is a hallmark of infant infection and major risk factor for fatal pertussis. Taken together our data demonstrates that α1AT is a novel PT inhibitor and that further evaluation and development of α1AT as a therapeutic agent for pertussis is warranted. Importantly, purified α1AT is already in use clinically as an intravenous augmentation therapy for those with genetic α1AT deficiency and could be repurposed to clinical management of pertussis.

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.

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.

IL-17 and IFN-γ-producing Respiratory Tissue-Resident Memory CD4 T Cells Persist for Decades in Adults Immunized as Children With Whole-Cell Pertussis Vaccines.

The objective was to determine if antigen-specific tissue-resident memory T (TRM) cells persist in respiratory tissues of adults immunized as children with whole-cell pertussis (wP) or acellular pertussis (aP) vaccines. Mononuclear cells from tonsil or nasal tissue cells were cultured with Bordetella pertussis antigens and TRM cells quantified by flow cytometry. Adults immunized with wP vaccines as children had significantly more interleukin 17A (IL-17A) and interferon-γ (IFN-γ)-producing TRM cells that respond to B. pertussis antigens in respiratory tissues when compared with aP-primed donors. Our findings demonstrate that wP vaccines induce CD4 TRM cells that can persist in respiratory tissues for decades.

Role of the Putative Histidine Kinase BP1092 in Bordetella pertussis Virulence Regulation and Intracellular Survival.

Bordetella pertussis persists inside host cells, and virulence factors are crucial for intracellular adaptation. The regulation of B. pertussis virulence factor transcription primarily occurs through the modulation of the two-component system (TCS) known as BvgAS. However, additional regulatory systems have emerged as potential contributors to virulence regulation. Here, we investigate the impact of BP1092, a putative TCS histidine kinase that shows increased levels after bacterial internalization by macrophages, on B. pertussis proteome adaptation under nonmodulating (Bvg+) and modulating (Bvg-) conditions. Using mass spectrometry, we compare B. pertussis wild-type (wt), a BP1092-deficient mutant (ΔBP1092), and a ΔBP1092 trans-complemented strain under both conditions. We find an altered abundance of 10 proteins, including five virulence factors. Specifically, under nonmodulating conditions, the mutant strain showed decreased levels of FhaB, FhaS, and Cya compared to the wt. Conversely, under modulating conditions, the mutant strain exhibited reduced levels of BvgA and BvgS compared to those of the wt. Functional assays further revealed that the deletion of BP1092 gene impaired B. pertussis ability to survive within human macrophage THP-1 cells. Taken together, our findings allow us to propose BP1092 as a novel player involved in the intricate regulation of B. pertussis virulence factors and thus in adaptation to the intracellular environment. The data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD041940.

Virus-like particles displaying the mature C-terminal domain of filamentous hemagglutinin are immunogenic and protective against Bordetella pertussis respiratory infection in mice.

Bordetella pertussis, the bacterium responsible for whooping cough, remains a significant public health challenge despite the existing licensed pertussis vaccines. Current acellular pertussis vaccines, though having favorable reactogenicity and efficacy profiles, involve complex and costly production processes. In addition, acellular vaccines have functional challenges such as short-lasting duration of immunity and limited antigen coverage. Filamentous hemagglutinin (FHA) is an adhesin of B. pertussis that is included in all multivalent pertussis vaccine formulations. Antibodies to FHA have been shown to prevent bacterial attachment to respiratory epithelial cells, and T cell responses to FHA facilitate cell-mediated immunity. In this study, FHA's mature C-terminal domain (MCD) was evaluated as a novel vaccine antigen. MCD was conjugated to virus-like particles via SpyTag-SpyCatcher technology. Prime-boost vaccine studies were performed in mice to characterize immunogenicity and protection against the intranasal B. pertussis challenge. MCD-SpyVLP was more immunogenic than SpyTag-MCD antigen alone, and in Tohama I strain challenge studies, improved protection against challenge was observed in the lungs at day 3 and in the trachea and nasal wash at day 7 post-challenge. Furthermore, a B. pertussis strain encoding genetically inactivated pertussis toxin was used to evaluate MCD-SpyVLP vaccine immunity. Mice vaccinated with MCD-SpyVLP had significantly lower respiratory bacterial burden at both days 3 and 7 post-challenge compared to mock-vaccinated animals. Overall, these data support the use of SpyTag-SpyCatcher VLPs as a platform for use in vaccine development against B. pertussis and other pathogens.

Immunization with an mRNA DTP vaccine protects against pertussis in rats.

Bordetella pertussis is a Gram-negative bacterium that is the causative agent of the respiratory disease known as pertussis. Since the switch to the acellular vaccines of DTaP and Tap, pertussis cases in the US have risen and cyclically fallen. We have observed that mRNA pertussis vaccines are immunogenic and protective in mice. Here, we further evaluated the pertussis toxoid mRNA antigen and refined the formulation based on optimal pertussis toxin neutralization in vivo. We next evaluated the mRNA pertussis vaccine in Sprague-Dawley rats using an aerosol B. pertussis challenge model paired with whole-body plethysmography to monitor coughing and respiratory function. Female Sprague-Dawley rats were primed and boosted with either commercially available vaccines (DTaP or wP-DTP), an mRNA-DTP vaccine, or mock-vaccinated. The mRNA-DTP vaccine was immunogenic in rats and induced antigen-specific IgG antibodies comparable to DTaP. Rats were then aerosol challenged with a streptomycin-resistant emerging clinical isolate D420Sm1. Bacterial burden was assessed at days 1 and 9 post-challenge, and the mRNA vaccine reduced burden equal to both DTaP and wP-DTP. Whole-body plethysmography revealed that mRNA-DTP vaccinated rats were well protected against coughing which was comparable to the non-challenged group. These data suggest that an mRNA-DTP vaccine is immunogenic in rats and provides protection against aerosolized B. pertussis challenge in Sprague-Dawley rats.

BECC438b TLR4 agonist supports unique immune response profiles from nasal and muscular DTaP pertussis vaccines in murine challenge models.

The protection afforded by acellular pertussis vaccines wanes over time, and there is a need to develop improved vaccine formulations. Options to improve the vaccines involve the utilization of different adjuvants and administration via different routes. While intramuscular (IM) vaccination provides a robust systemic immune response, intranasal (IN) vaccination theoretically induces a localized immune response within the nasal cavity. In the case of a Bordetella pertussis infection, IN vaccination results in an immune response that is similar to natural infection, which provides the longest duration of protection. Current acellular formulations utilize an alum adjuvant, and antibody levels wane over time. To overcome the current limitations with the acellular vaccine, we incorporated a novel TLR4 agonist, BECC438b, into both IM and IN acellular formulations to determine its ability to protect against infection in a murine airway challenge model. Following immunization and challenge, we observed that DTaP + BECC438b reduced bacterial burden within the lung and trachea for both administration routes when compared with mock-vaccinated and challenged (MVC) mice. Interestingly, IN administration of DTaP + BECC438b induced a Th1-polarized immune response, while IM vaccination polarized toward a Th2 immune response. RNA sequencing analysis of the lung demonstrated that DTaP + BECC438b activates biological pathways similar to natural infection. Additionally, IN administration of DTaP + BECC438b activated the expression of genes involved in a multitude of pathways associated with the immune system. Overall, these data suggest that BECC438b adjuvant and the IN vaccination route can impact efficacy and responses of pertussis vaccines in pre-clinical mouse models.

ER-transiting bacterial toxins amplify STING innate immune responses and elicit ER stress.

The cGAS/STING sensor system drives innate immune responses to intracellular microbial double-stranded DNA (dsDNA) and bacterial cyclic nucleotide second messengers (e.g., c-di-AMP). STING-dependent cell-intrinsic responses can increase resistance to microbial infection and speed pathogen clearance. Correspondingly, STING activation and signaling are known to be targeted for suppression by effectors from several bacterial pathogens. Whether STING responses are also positively regulated through sensing of specific bacterial effectors is less clear. We find that STING activation through dsDNA, by its canonical ligand 2'-3' cGAMP, or the small molecule DMXAA is potentiated following intracellular delivery of the AB5 toxin family member pertussis toxin from Bordetella pertussis or the B subunit of cholera toxin from Vibrio cholerae. Entry of pertussis toxin or cholera toxin B into mouse macrophages triggers markers of endoplasmic reticulum (ER) stress and enhances ligand-dependent STING responses at the level of STING receptor activation in a manner that is independent of toxin enzymatic activity. Our results provide an example in which STING responses integrate information about the presence of relevant ER-transiting bacterial toxins into the innate inflammatory response and may help to explain the in vivo adjuvant effects of catalytically inactive toxins.

Eosinophils as drivers of bacterial immunomodulation and persistence.

Traditionally, eosinophils have been linked to parasitic infections and pathological disease states. However, emerging literature has unveiled a more nuanced and intricate role for these cells, demonstrating their key functions in maintaining mucosal homeostasis. Eosinophils exhibit diverse phenotypes and exert multifaceted effects during infections, ranging from promoting pathogen persistence to triggering allergic reactions. Our investigations primarily focus on Bordetella spp., with particular emphasis on Bordetella bronchiseptica, a natural murine pathogen that induces diseases in mice akin to pertussis in humans. Recent findings from our published work have unveiled a striking interaction between B. bronchiseptica and eosinophils, facilitated by the btrS-mediated mechanism. This interaction serves to enhance pathogen persistence while concurrently delaying adaptive immune responses. Notably, this role of eosinophils is only noted in the absence of a functional btrS signaling pathway, indicating that wild-type B. bronchiseptica, and possibly other Bordetella spp., possess such adeptness in manipulating eosinophils that the true function of these cells remains obscured during infection. In this review, we present the mounting evidence pointing toward eosinophils as targets of bacterial exploitation, facilitating pathogen persistence and fostering chronic infections in diverse mucosal sites, including the lungs, gut, and skin. We underscore the pivotal role of the master regulator of Bordetella pathogenesis, the sigma factor BtrS, in orchestrating eosinophil-dependent immunomodulation within the context of pulmonary infection. These putative convergent strategies of targeting eosinophils offer promising avenues for the development of novel therapeutics targeting respiratory and other mucosal pathogens.

Assessing the Impact of the 2020 Council of State and Territorial Epidemiologists Case Definition for Pertussis on Reported Pertussis Cases.

BACKGROUND: In 2020, the Council of State and Territorial Epidemiologists (CSTE) pertussis case definition was modified; the main change was classifying polymerase chain reaction (PCR)-positive cases as confirmed, regardless of cough duration. Pertussis data reported through Enhanced Pertussis Surveillance (EPS) in 7 sites and the National Notifiable Diseases Surveillance System (NNDSS) were used to evaluate the impact of the new case definition. METHODS: We compared the number of EPS cases with cough onset in 2020 to the number that would have been reported based on the prior (2014) CSTE case definition. To assess the impact of the change nationally, the proportion of EPS cases newly reportable under the 2020 CSTE case definition was applied to 2020 NNDSS data to estimate how many additional cases were captured nationally. RESULTS: Among 442 confirmed and probable cases reported to EPS states in 2020, 42 (9.5%) were newly reportable according to the 2020 case definition. Applying this proportion to the 6124 confirmed and probable cases reported nationally in 2020, we estimated that the new definition added 582 cases. Had the case definition not changed, reported cases in 2020 would have decreased by 70% from 2019; the observed decrease was 67%. CONCLUSIONS: Despite a substantial decrease in reported pertussis cases in the setting of coronavirus disease 2019 (COVID-19), our data show that the 2020 pertussis case definition change resulted in additional case reporting compared with the previous case definition, providing greater opportunities for public health interventions such as prophylaxis of close contacts.

Prevalence of Pertactin-Deficient Bordetella pertussis Isolates, Slovenia.

In Slovenia, primary acellular pertussis vaccines (ACVs) containing pertactin (PRN) were mostly used during 1999-2016; ACVs without PRN were introduced in 2017. Among 123 Bordetella pertussis strains collected during 2002-2020, a total of 48 were PRN-deficient; 44 were collected after 2017. Changes to ACVs could increase PRN-deficient B. pertussis and infections.

Population-Based Study of Pertussis Incidence and Risk Factors among Persons >50 Years of Age, Australia.

Despite vaccination programs, pertussis has been poorly controlled, especially among older adults in Australia. This longitudinal, retrospective, observational study aimed to estimate the incidence and risk factors of pertussis among persons ≥50 years of age in Australia in the primary care setting, including those with underlying chronic obstructive pulmonary disease (COPD) or asthma. We used the IQVIA general practitioner electronic medical record database to identify patients ≥50 years of age with a clinical diagnosis of pertussis during 2015-2019. Pertussis incidence rates ranged from 57.6 to 91.4 per 100,000 persons and were higher among women and highest in those 50-64 years of age. Patients with COPD or asthma had higher incidence rates and an increased risk for pertussis compared with the overall population ≥50 years of age. Our findings suggest that persons ≥50 years of age in Australia with COPD or asthma have a higher incidence of and risk for pertussis diagnosis.

Reemergence of Bordetella parapertussis, United States, 2019-2023.

To determine changes in Bordetella pertussis and B. parapertussis detection rates, we analyzed 1.43 million respiratory multiplex PCR test results from US facilities from 2019 through mid-2023. From mid-2022 through mid-2023, Bordetella spp. detection increased 8.5-fold; 95% of detections were B. parapertussis. While B. parapertussis rates increased, B. pertussis rates decreased.

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.

Bystander activation of tissue-resident memory CD4 T cells: Getting by with a little help from unfamiliar T-cell friends.

Reexposure to a pathogen triggers the activation of memory T cells that have already encountered a similar microbe. These long-lived CD4 T cells either circulate through the blood and tissues or reside within organs and are referred to as tissue-resident T cells (CD4 TRM ). In the current issue of the European Journal of Immunology [Eur. J. Immunol. 2023. 53: 2250247] issue, Curham et al. found that tissue-resident memory CD4 T cells in the lung and nasal tissues can respond to noncognate immune challenges. CD4 TRM cells, which were formed in response to Bordetella pertussis, proliferated and produced IL-17A in response to a secondary challenge with heat-killed Klebsiella pneumonia or lipopolysaccharide (LPS). This bystander response depends on the presence of dendritic cells that provide inflammatory cytokines. Furthermore, post K. pneumonia, intranasal immunization with whole cell pertussis vaccine reduced bacterial burden in the nasal tissue in a CD4 T-cell-dependent manner. The study indicates that the noncognate activation of TRM may serve as an innate-like immune response that rapidly develops before establishing a new pathogen-specific adaptive immune response.

Strengthening Bordetella pertussis genomic surveillance by direct sequencing of residual positive specimens.

Whole-genome sequencing (WGS) of microbial pathogens recovered from patients with infectious disease facilitates high-resolution strain characterization and molecular epidemiology. However, increasing reliance on culture-independent methods to diagnose infectious diseases has resulted in few isolates available for WGS. Here, we report a novel culture-independent approach to genome characterization of Bordetella pertussis, the causative agent of pertussis and a paradigm for insufficient genomic surveillance due to limited culture of clinical isolates. Sequencing libraries constructed directly from residual pertussis-positive diagnostic nasopharyngeal specimens were hybridized with biotinylated RNA "baits" targeting B. pertussis fragments within complex mixtures that contained high concentrations of host and microbial background DNA. Recovery of B. pertussis genome sequence data was evaluated with mock and pooled negative clinical specimens spiked with reducing concentrations of either purified DNA or inactivated cells. Targeted enrichment increased the yield of B. pertussis sequencing reads up to 90% while simultaneously decreasing host reads to less than 10%. Filtered sequencing reads provided sufficient genome coverage to perform characterization via whole-genome single nucleotide polymorphisms and whole-genome multilocus sequencing typing. Moreover, these data were concordant with sequenced isolates recovered from the same specimens such that phylogenetic reconstructions from either consistently clustered the same putatively linked cases. The optimized protocol is suitable for nasopharyngeal specimens with diagnostic IS481 Ct < 35 and >10 ng DNA. Routine implementation of these methods could strengthen surveillance and study of pertussis resurgence by capturing additional cases with genomic characterization.

Use and Waste of Reconstituted Whole Blood Exchange Transfusions: An 11-year National Observational Study.

OBJECTIVES: To identify indications for exchange transfusions, assess the use and waste of exchange transfusion products (ie, reconstituted whole blood exchange transfusions), and determine nationwide distribution and prevalence of these transfusions in the Netherlands. STUDY DESIGN: All 9 neonatal intensive care units and 15 non-neonatal intensive care unit hospitals participated in this retrospective, observational, cohort study. We retrieved data on the indications for and use of all exchange transfusion products ordered by participating centers over an 11-year period. RESULTS: A total of 574 patients for whom 1265 products were ordered were included for analyses. Severe ABO (32.6%) and non-ABO (25.2%) immune hemolysis and subsequent hyperbilirubinemia were the most frequent indications. Rare indications were severe leukocytosis in Bordetella pertussis (2.1%) and severe anemia (1.5%). Approximately one-half of all ordered products remained unused. In 278 of 574 neonates (48.4%), ≥1 products were not used, of which 229 (82.7%) were due to the resolving of severe hyperbilirubinemia with further intensification of phototherapy. The overall prevalence of neonates who received an exchange transfusion was 14.6:100 000 liveborn neonates. CONCLUSIONS: A considerable proportion of products remained unused, and annually a limited number of patients are treated with an exchange transfusion in the Netherlands, highlighting the rarity of the procedure in the Netherlands.

Chapter Controlled Human Infection with Bordetella pertussis.

Bordetella pertussis, a slow-growing Gram-negative coccobacillus and the causative agent of whooping cough, is one of the leading causes of vaccine-preventable death and morbidity globally. A state of asymptomatic human carriage has not yet been demonstrated by population studies but is likely to be an important reservoir for community transmission of infection. Such a carriage state may be a target for future vaccine strategies. This chapter presents a short summary of the characteristics of B. pertussis, which should be taken into account when developing a human challenge model and any future experimental medicine interventions. Three studies involving deliberate infection with B. pertussis have been described to date. The first of these was a scientifically and ethically unacceptable paediatric challenge study involving four children in 1930. The second was an investigation of a putative live vaccine using a genetically modified and attenuated strain of B. pertussis. Finally, a systematically constructed human challenge model using a wild-type, potentially pathogenic strain has been established. The latter study has demonstrated that deliberate induction of asymptomatic colonisation in humans is safe and immunogenic, with colonised participants exhibiting seroconversion to pertussis antigens. It has also shown nasal wash to be a more sensitive method of detecting the presence of B. pertussis than either pernasal swab or throat swab, and that B. pertussis carriage can be cleared effectively with Azithromycin. The development of this wild-type B. pertussis human challenge model will allow the investigation of host-pathogen and facilitate future vaccine development.

Synthesis of oligosaccharides from terminal B. pertussis LPS pentasaccharide and definition of the minimal epitope recognized by anti-pertussis antibodies.

Pertussis vaccines have been very effective in controlling whooping-cough epidemics but are ineffective in controlling circulation in older children and adults, thus facilitating the onset of future outbreaks. Antibodies against the lipopolysaccharide could reduce the carriage of the bacteria, its circulation, and transmission. The oligosaccharide fragments from the lipopolysaccharide may become a potential complement to existing vaccines in the form of protein glycoconjugates. An important step in the development of this type of vaccine is defining the minimal oligosaccharide epitope recognized by B. pertussis anti-lipopolysaccharide antibodies. This paper describes the complete synthesis of oligosaccharides containing two to five monosaccharide units corresponding to the pentasaccharide at the nonreducing end of the lipooligosaccharide and their recognition by mice and rabbit antibodies elicited against whole-cell B. pertussis. For the first time, we report that the terminal disaccharide, α-D-GlcNAcp-(1 → 4)-(2,3-di-NAc)-D-ManAp acid is the minimal structure recognized by antibodies induced by B. pertussis.