Genomic dissection of the microevolution of Australian epidemic Bordetella pertussis.

ABSTRACTWhooping cough (pertussis) is a highly contagious respiratory disease caused by the bacterium Bordetella pertussis. Despite high vaccine coverage, pertussis has re-emerged in many countries including Australia and caused two large epidemics in Australia since 2007. Here, we undertook a genomic and phylogeographic study of 385 Australian B. pertussis isolates collected from 2008 to 2017. The Australian B. pertussis population was found to be composed of mostly ptxP3 strains carrying different fim3 alleles, with ptxP3-fim3A genotype expanding far more than ptxP3-fim3B. Within the former, there were six co-circulating epidemic lineages (EL1 to EL6). The multiple ELs emerged, expanded, and then declined at different time points over the two epidemics. In population genetics terms, both hard and soft selective sweeps through vaccine selection pressures have determined the population dynamics of Australian B. pertussis. Relative risk estimation suggests that once a new B. pertussis lineage emerged, it was more likely to spread locally within the first 1.5 years. However, after 1.5 years, any new lineage was likely to expand to a wider region. Phylogenetic analysis revealed the expansion of ptxP3 strains was also associated with replacement of the type III secretion system allele bscI1 with bscI3. bscI3 is associated with decreased T3SS secretion and may allow B. pertussis to reduce immune recognition. This study advanced our understanding of the epidemic population structure and spatial and temporal dynamics of B. pertussis in a highly immunized population.

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.

Bordetella pertussis pertactin knock-out strains reveal immunomodulatory properties of this virulence factor.

Whooping cough, caused by Bordetella pertussis, has resurged and presents a global health burden worldwide. B. pertussis strains unable to produce the acellular pertussis vaccine component pertactin (Prn), have been emerging and in some countries represent up to 95% of recent clinical isolates. Knowledge on the effect that Prn deficiency has on infection and immunity to B. pertussis is crucial for the development of new strategies to control this disease. Here, we characterized the effect of Prn production by B. pertussis on human and murine dendritic cell (DC) maturation as well as in a murine model for pertussis infection. We incubated human monocyte-derived DCs (moDCs) with multiple isogenic Prn knockout (Prn-KO) and corresponding parental B. pertussis strains constructed either in laboratory reference strains with a Tohama I background or in a recently circulating clinical isolate. Results indicate that, compared to the parental strains, Prn-KO strains induced an increased production of pro-inflammatory cytokines by moDCs. This pro-inflammatory phenotype was also observed upon stimulation of murine bone marrow-derived DCs. Moreover, RNA sequencing analysis of lungs from mice infected with B. pertussis Prn-KO revealed increased expression of genes involved in cell death. These in vitro and in vivo findings indicate that B. pertussis strains which do not produce Prn induce a stronger pro-inflammatory response and increased cell death upon infection, suggesting immunomodulatory properties for Prn.

Adaptation of Bordetella pertussis to the Respiratory Tract.

There is a lack of insight into the basic mechanisms by which Bordetella pertussis adapts to the local host environment during infection. We analyzed B. pertussis gene expression in the upper and lower airways of mice and compared this to SO4-induced in vitro Bvg-regulated gene transcription. Approximately 30% of all genes were differentially expressed between in vitro and in vivo conditions. This included several novel potential vaccine antigens that were exclusively expressed in vivo. Significant differences in expression profile and metabolic pathways were identified between the upper versus the lower airways, suggesting distinct antigenic profiles. We found high-level expression of several Bvg-repressed genes during infection, and mouse vaccination experiments using purified protein fractions from both Bvg- and Bvg+ cultures demonstrated protection against intranasal B. pertussis challenge. This study provides novel insights into the in vivo adaptation of B. pertussis and may facilitate the improvement of pertussis vaccines.

Construction and evaluation of Bordetella pertussis live attenuated vaccine strain BPZE1 producing Fim3.

Pertussis or whooping cough is currently the most prevalent vaccine-preventable childhood disease despite >85% global vaccination coverage. In recent years incidence has greatly increased in several high-income countries that have switched from the first-generation, whole-cell vaccine to the newer acellular vaccines, calling for improved vaccination strategies with better vaccines. We have developed a live attenuated pertussis vaccine candidate, called BPZE1, which is currently in clinical development. Unlike other pertussis vaccines, BPZE1 has been shown to provide strong protection against infection by the causative agent of pertussis, Bordetella pertussis, in non-human primates. BPZE1 is a derivative of the B. pertussis strain Tohama I, which produces serotype 2 (Fim2) but not serotype 3 fimbriae (Fim3). As immune responses to fimbriae are likely to contribute to protection, we constructed a BPZE1 derivative, called BPZE1f3, that produces both serotypes of fimbriae. Whereas nasal vaccination of mice with BPZE1 induced antibodies to Fim2 but not to Fim3, vaccination with BPZE1f3 elicited antibodies to both Fim2 and Fim3 at approximately the same level. In mice, both BPZE1 and BPZE1f3 provided equal levels of protection against clinical isolates that either produce Fim2 alone, both Fim2 and Fim3, or no fimbriae. However, vaccination with BPZE1f3 provided significantly stronger protection against Fim3-only producing B. pertussis than vaccination with BPZE1, indicating that immune responses to fimbriae contribute to serotype-specific protection against B. pertussis infection.

Bordetella Pertussis virulence factors in the continuing evolution of whooping cough vaccines for improved performance.

Despite high vaccine coverage, whooping cough caused by Bordetella pertussis remains one of the most common vaccine-preventable diseases worldwide. Introduction of whole-cell pertussis (wP) vaccines in the 1940s and acellular pertussis (aP) vaccines in 1990s reduced the mortality due to pertussis. Despite induction of both antibody and cell-mediated immune (CMI) responses by aP and wP vaccines, there has been resurgence of pertussis in many countries in recent years. Possible reasons hypothesised for resurgence have ranged from incompliance with the recommended vaccination programmes with the currently used aP vaccine to infection with a resurged clinical isolates characterised by mutations in the virulence factors, resulting in antigenic divergence with vaccine strain, and increased production of pertussis toxin, resulting in dampening of immune responses. While use of these vaccines provide varying degrees of protection against whooping cough, protection against infection and transmission appears to be less effective, warranting continuation of efforts in the development of an improved pertussis vaccine formulations capable of achieving this objective. Major approaches currently under evaluation for the development of an improved pertussis vaccine include identification of novel biofilm-associated antigens for incorporation in current aP vaccine formulations, development of live attenuated vaccines and discovery of novel non-toxic adjuvants capable of inducing both antibody and CMI. In this review, the potential roles of different accredited virulence factors, including novel biofilm-associated antigens, of B. pertussis in the evolution, formulation and delivery of improved pertussis vaccines, with potential to block the transmission of whooping cough in the community, are discussed.

Emerging Bordetella pertussis Strains Induce Enhanced Signaling of Human Pattern Recognition Receptors TLR2, NOD2 and Secretion of IL-10 by Dendritic Cells.

Vaccines against pertussis have been available for more than 60 years. Nonetheless, this highly contagious disease is reemerging even in countries with high vaccination coverage. Genetic changes of Bordetella pertussis over time have been suggested to contribute to the resurgence of pertussis, as these changes may favor escape from vaccine-induced immunity. Nonetheless, studies on the effects of these bacterial changes on the immune response are limited. Here, we characterize innate immune recognition and activation by a collection of genetically diverse B. pertussis strains isolated from Dutch pertussis patients before and after the introduction of the pertussis vaccines. For this purpose, we used HEK-Blue cells transfected with human pattern recognition receptors TLR2, TLR4, NOD2 and NOD1 as a high throughput system for screening innate immune recognition of more than 90 bacterial strains. Physiologically relevant human monocyte derived dendritic cells (moDC), purified from peripheral blood of healthy donors were also used. Findings indicate that, in addition to inducing TLR2 and TLR4 signaling, all B. pertussis strains activate the NOD-like receptor NOD2 but not NOD1. Furthermore, we observed a significant increase in TLR2 and NOD2, but not TLR4, activation by strains circulating after the introduction of pertussis vaccines. When using moDC, we observed that the recently circulating strains induced increased activation of these cells with a dominant IL-10 production. In addition, we observed an increased expression of surface markers including the regulatory molecule PD-L1. Expression of PD-L1 was decreased upon blocking TLR2. These in vitro findings suggest that emerging B. pertussis strains have evolved to dampen the vaccine-induced inflammatory response, which would benefit survival and transmission of this pathogen. Understanding how this disease has resurged in a highly vaccinated population is crucial for the design of improved vaccines against pertussis.

Highly differentiated human airway epithelial cells: a model to study host cell-parasite interactions in pertussis.

BACKGROUND: Bordetella pertussis colonizes the human respiratory mucosa. Most studies on B. pertussis adherence have relied on cultured mammalian cells that lack key features present in differentiated human airway cells or on animal models that are not natural hosts of B. pertussis. The objectives of this work were to evaluate B. pertussis infection in highly differentiated human airway cells in vitro and to show the role of B. pertussis fimbriae in cell adherence. METHODS: Primary human airway epithelial (PHAE) cells from human bronchi and a human bronchial epithelial (HBE) cell line were grown in vitro under air-liquid interface conditions. RESULTS: PHAE and HBE cells infected with B. pertussis wild-type strain revealed bacterial adherence to the apical surface of cells, bacteria-induced cytoskeleton changes, and cell detachment. Mutations in the major fimbrial subunits Fim2/3 or in the minor fimbrial adhesin subunit FimD affected B. pertussis adherence to predominantly HBE cells. This cell model recapitulates the morphologic features of the human airway infected by B. pertussis and confirms the role of fimbriae in B. pertussis adherence. Furthermore, HBE cells show that fimbrial subunits, and specifically FimD adhesin, are critical in B. pertussis adherence to airway cells. CONCLUSIONS: The relevance of this model to study host-parasite interaction in pertussis lies in the striking physiologic and morphologic similarity between the PHAE and HBE cells and the human airway ciliated and goblet cells in vivo. These cells can proliferate in vitro, differentiate, and express the same genetic profile as human respiratory cells in vivo.

Bordetella pertussis Isolates from Argentinean Whooping Cough Patients Display Enhanced Biofilm Formation Capacity Compared to Tohama I Reference Strain.

Pertussis is a highly contagious disease mainly caused by Bordetella pertussis. Despite the massive use of vaccines, since the 1950s the disease has become re-emergent in 2000 with a shift in incidence from infants to adolescents and adults. Clearly, the efficacy of current cellular or acellular vaccines, formulated from bacteria grown in stirred bioreactors is limited, presenting a challenge for future vaccine development. For gaining insights into the role of B. pertussis biofilm development for host colonization and persistence within the host, we examined the biofilm forming capacity of eight argentinean clinical isolates recovered from 2001 to 2007. All clinical isolates showed an enhanced potential for biofilm formation compared to the reference strain Tohama I. We further selected the clinical isolate B. pertussis 2723, exhibiting the highest biofilm biomass production, for quantitative proteomic profiling by means of two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) coupled with mass spectrometry, which was accompanied by targeted transcriptional analysis. Results revealed an elevated expression of several virulence factors, including adhesins involved in biofilm development. In addition, we observed a higher expression of energy metabolism enzymes in the clinical isolate compared to the Tohama I strain. Furthermore, all clinical isolates carried a polymorphism in the bvgS gene. This mutation was associated to an increased sensitivity to modulation and a faster rate of adhesion to abiotic surfaces. Thus, the phenotypic biofilm characteristics shown by the clinical isolates might represent an important, hitherto underestimated, adaptive strategy for host colonization and long time persistence within the host.

Complete Genome Sequences of 11 Bordetella pertussis Strains Representing the Pandemic ptxP3 Lineage.

Pathogen adaptation has contributed to the resurgence of pertussis. To facilitate our understanding of this adaptation we report here 11 completely closed and annotated Bordetella pertussis genomes representing the pandemic ptxP3 lineage. Our analyses included six strains which do not produce the vaccine components pertactin and/or filamentous hemagglutinin.

Laboratory Diagnosis of Pertussis.

The introduction of vaccination in the 1950s significantly reduced the morbidity and mortality of pertussis. However, since the 1990s, a resurgence of pertussis has been observed in vaccinated populations, and a number of causes have been proposed for this phenomenon, including improved diagnostics, increased awareness, waning immunity, and pathogen adaptation. The resurgence of pertussis highlights the importance of standardized, sensitive, and specific laboratory diagnoses, the lack of which is responsible for the large differences in pertussis notifications between countries. Accurate laboratory diagnosis is also important for distinguishing between the several etiologic agents of pertussis-like diseases, which involve both viruses and bacteria. If pertussis is diagnosed in a timely manner, antibiotic treatment of the patient can mitigate the symptoms and prevent transmission. During an outbreak, timely diagnosis of pertussis allows prophylactic treatment of infants too young to be (fully) vaccinated, for whom pertussis is a severe, sometimes fatal disease. Finally, reliable diagnosis of pertussis is required to reveal trends in the (age-specific) disease incidence, which may point to changes in vaccine efficacy, waning immunity, and the emergence of vaccine-adapted strains. Here we review current approaches to the diagnosis of pertussis and discuss their limitations and strengths. In particular, we emphasize that the optimal diagnostic procedure depends on the stage of the disease, the age of the patient, and the vaccination status of the patient.

Genetic Variation of Bordetella pertussis in Austria.

In Austria, vaccination coverage against Bordetella pertussis infections during infancy is estimated at around 90%. Within the last years, however, the number of pertussis cases has increased steadily, not only in children but also in adolescents and adults, indicating both insufficient herd immunity and vaccine coverage. Waning immunity in the host and/or adaptation of the bacterium to the immunised hosts could contribute to the observed re-emergence of pertussis. In this study we therefore addressed the genetic variability in B. pertussis strains from several Austrian cities. Between the years 2002 and 2008, 110 samples were collected from Vienna (n = 32), Linz (n = 63) and Graz (n = 15) by nasopharyngeal swabs. DNA was extracted from the swabs, and bacterial sequence polymorphisms were examined by MLVA (multiple-locus variable number of tandem repeat analysis) (n = 77), by PCR amplification and conventional Sanger sequencing of the polymorphic regions of the prn (pertactin) gene (n = 110), and by amplification refractory mutation system quantitative PCR (ARMS-qPCR) (n = 110) to directly address polymorphisms in the genes encoding two pertussis toxin subunits (ptxA and ptxB), a fimbrial adhesin (fimD), tracheal colonisation factor (tcfA), and the virulence sensor protein (bvgS). Finally, the ptxP promoter region was screened by ARMS-qPCR for the presence of the ptxP3 allele, which has been associated with elevated production of pertussis toxin. The MLVA analysis revealed the highest level of polymorphisms with an absence of MLVA Type 29, which is found outside Austria. Only Prn subtypes Prn1/7, Prn2 and Prn3 were found with a predominance of the non-vaccine type Prn2. The analysis of the ptxA, ptxB, fimD, tcfA and bvgS polymorphisms showed a genotype mixed between the vaccine strain Tohama I and a clinical isolate from 2006 (L517). The major part of the samples (93%) displayed the ptxP3 allele. The consequences for the vaccination strategy are discussed.

Seroprevalence of pertussis in the Gambia: evidence for continued circulation of bordetella pertussis despite high vaccination rates.

BACKGROUND: Bordetella pertussis can cause severe respiratory disease and death in children. In recent years, large outbreaks have occurred in high-income countries; however, little is known about pertussis incidence in sub-Saharan Africa. METHODS: We evaluated antibody responses to pertussis toxin (Ptx) from individuals aged between 2 and 90 years in rural Gambia. IgG-Ptx was measured using luminex xMAP technology. IgG-Ptx geometric mean concentrations (GMC) and their 95% confidence intervals were calculated. The proportion seropositive (>20 EU/mL or ≥62.5 EU/mL) and GMCs were compared by age, sex, ethnic group, vaccination status, birth order and number of siblings per household using logistic and linear regression. RESULTS: 76.3% had anti-Ptx levels <20 EU/mL, 17.5% had concentrations between 20 and 62.5 EU/mL, 4.4% had concentrations between 62.5 and 125 EU/mL and 1.8% had concentrations ≥125 EU/mL. The overall Ptx antibody GMC was 6.4 EU/mL (95% confidence interval: 5.8-6.9). Higher antibody concentrations were observed in older populations with evidence for an increase in infection risk with increasing age (1.9% yearly increase, 95% confidence interval: 1.3-2.5). No child under 6 years of age had GMC above 62.5 EU/mL but 29.5% had concentrations between 20 and 62.5 EU/mL. CONCLUSIONS: These data provide evidence that B. pertussis is being transmitted within this population despite high vaccination coverage. Re-infection may occur implying that immunity from childhood vaccination may not be lifelong. In the absence of data on actual clinical cases of pertussis, seroprevalence studies remain valuable tools to assess the transmission dynamics of B. pertussis.

Complement evasion by Bordetella pertussis: implications for improving current vaccines.

Bordetella pertussis causes whooping cough or pertussis, a highly contagious disease of the respiratory tract. Despite high vaccination coverage, reported cases of pertussis are rising worldwide and it has become clear that the current vaccines must be improved. In addition to the well-known protective role of antibodies and T cells during B. pertussis infection, innate immune responses such as the complement system play an essential role in B. pertussis killing. In order to evade this complement activation and colonize the human host, B. pertussis expresses several molecules that inhibit complement activation. Interestingly, one of the known complement evasion proteins, autotransporter Vag8, is highly expressed in the recently emerged B. pertussis isolates. Here, we describe the current knowledge on how B. pertussis evades complement-mediated killing. In addition, we compare this to complement evasion strategies used by other bacterial species. Finally, we discuss the consequences of complement evasion by B. pertussis on adaptive immunity and how identification of the bacterial molecules and the mechanisms involved in complement evasion might help improve pertussis vaccines.

Studying Bordetella pertussis populations by use of SNPeX, a simple high-throughput single nucleotide polymorphism typing method.

Large outbreaks of pertussis occur despite vaccination. A first step in the analyses of outbreaks is strain typing. However, the typing of Bordetella pertussis, the causative agent of pertussis, is problematic because the available assays are insufficiently discriminatory, not unequivocal, time-consuming, and/or costly. Here, we describe a single nucleotide primer extension assay for the study of B. pertussis populations, SNPeX (single nucleotide primer extension), which addresses these problems. The assay is based on the incorporation of fluorescently labeled dideoxynucleotides (ddNTPs) at the 3' end of allele-specific poly(A)-tailed primers and subsequent analysis with a capillary DNA analyzer. Each single nucleotide polymorphism (SNP) primer has a specific length, and as a result, up to 20 SNPs can be determined in one SNPeX reaction. Importantly, PCR amplification of target DNA is not required. We selected 38 SNPeX targets from the whole-genome sequencing data of 74 B. pertussis strains collected from across the world. The SNPeX-based phylogenetic trees preserved the general tree topology of B. pertussis populations based on whole-genome sequencing, with a minor loss of details. We envisage a strategy whereby SNP types (SnpTs) are quickly identified with the SNPeX assay during an outbreak, followed by whole-genome sequencing (WGS) of a limited number of isolates representing predominant SnpTs and the incorporation of novel SNPs in the SNPeX assay. The flexibility of the SNPeX assay allows the method to evolve along with the pathogen, making it a promising method for studying outbreaks of B. pertussis and other pathogens.

Bordetella pertussis naturally occurring isolates with altered lipooligosaccharide structure fail to fully mature human dendritic cells.

Bordetella pertussis is a Gram-negative bacterium and the causative agent of whooping cough. Despite high vaccination coverage, outbreaks are being increasingly reported worldwide. Possible explanations include adaptation of this pathogen, which may interfere with recognition by the innate immune system. Here, we describe innate immune recognition and responses to different B. pertussis clinical isolates. By using HEK-Blue cells transfected with different pattern recognition receptors, we found that 3 out of 19 clinical isolates failed to activate Toll-like receptor 4 (TLR4). These findings were confirmed by using the monocytic MM6 cell line. Although incubation with high concentrations of these 3 strains resulted in significant activation of the MM6 cells, it was found to occur mainly through interaction with TLR2 and not through TLR4. When using live bacteria, these 3 strains also failed to activate TLR4 on HEK-Blue cells, and activation of MM6 cells or human monocyte-derived dendritic cells was significantly lower than activation induced by the other 16 strains. Mass spectrum analysis of the lipid A moieties from these 3 strains indicated an altered structure of this molecule. Gene sequence analysis revealed mutations in genes involved in lipid A synthesis. Findings from this study indicate that B. pertussis isolates that do not activate TLR4 occur naturally and that this phenotype may give this bacterium an advantage in tempering the innate immune response and establishing infection. Knowledge on the strategies used by this pathogen in evading the host immune response is essential for the improvement of current vaccines or for the development of new ones.

Complete Genome Sequences of Bordetella pertussis Isolates B1917 and B1920, Representing Two Predominant Global Lineages.

Bordetella pertussis is the causative agent of pertussis, a disease which has resurged despite vaccination. We report the complete, annotated genomes of isolates B1917 and B1920, representing two lineages predominating globally in the last 50 years. The B1917 lineage has been associated with the resurgence of pertussis in the 1990s.

Proteomics-identified Bvg-activated autotransporters protect against bordetella pertussis in a mouse model.

Pertussis is a highly infectious respiratory disease of humans caused by the bacterium Bordetella pertussis. Despite high vaccination coverage, pertussis has re-emerged globally. Causes for the re-emergence of pertussis include limited duration of protection conferred by acellular pertussis vaccines (aP) and pathogen adaptation. Pathogen adaptations involve antigenic divergence with vaccine strains, the emergence of strains which show enhanced in vitro expression of a number of virulence-associated genes and of strains that do not express pertactin, an important aP component. Clearly, the identification of more effective B. pertussis vaccine antigens is of utmost importance. To identify novel antigens, we used proteomics to identify B. pertussis proteins regulated by the master virulence regulatory system BvgAS in vitro. Five candidates proteins were selected and it was confirmed that they were also expressed in the lungs of naïve mice seven days after infection. The five proteins were expressed in recombinant form, adjuvanted with alum and used to immunize mice as stand-alone antigens. Subsequent respiratory challenge showed that immunization with the autotransporters Vag8 and SphB1 significantly reduced bacterial load in the lungs. Whilst these antigens induced strong opsonizing antibody responses, we found that none of the tested alum-adjuvanted vaccines - including a three-component aP - reduced bacterial load in the nasopharynx, suggesting that alternative immunological responses may be required for efficient bacterial clearance from the nasopharynx.