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The pertussis agent

Despite high vaccine coverage in developed countries, the incidence of pertussis has increased in recent decades, often leading to severe consequences for sensitive groups, including infants. For this reason, improving the efficacy of pertussis vaccines is critical, and the addition of new antigens is a leading strategy to achieve this goal.

Pertussis, caused by Bordetella pertussis, is a reemerging disease that can produce severe disease manifestations in infants, including pulmonary hypertension (PH). B. pertussis-induced PH is a major risk factor for infection-induced death, but the molecular mechanisms promoting PH are unknown and there is no effective treatment. We examined B. pertussis-induced PH in infant and adult mouse models of pertussis by Fulton index, right heart catheterization, or Doppler echocardiogram. Our results demonstrate that B. pertussis-induced PH is age related and dependent on the expression of pertussis toxin by the bacterium. Hence, pertussis toxin-targeting treatments may ameliorate PH and fatal infant infection.

Pertussis, mainly caused by Bordetella pertussis, is a severe respiratory disease that can be fatal, especially in young infants. Vaccines, massively implemented since the middle of the last century, have substantially reduced the pertussis incidence, but have not been able to fully control the disease. One of the shortcomings of current pertussis vaccines is their inability to prevent infection by and transmission of B. pertussis, in contrast to immunity following natural infection. We have developed the live attenuated nasal vaccine BPZE1 and have shown that it prevents both disease and B. pertussis infection in preclinical models. This vaccine is now in clinical development. However, the initial clinical studies have suggested that vaccine take is hampered by pre-existing antibodies to pertactin. Here, we have constructed a pertactin-deficient BPZE1 derivative called BPZE1P in order to overcome this limitation. BPZE1P colonized the murine respiratory tract as efficiently as BPZE1 and induced antibodies at levels similar to those elicited by BPZE1. In the presence of pre-existing antibodies induced by acellular pertussis vaccination, BPZE1P colonized the mouse respiratory tract more efficiently than BPZE1. Both vaccines protected equally well the murine lungs and noses from challenge with laboratory and clinical strains of B. pertussis, including pertactin-deficient strains, against which current acellular pertussis vaccines are less efficient. BPZE1P may thus be an interesting alternative to BPZE1 to overcome vaccine take limitations due to pre-existing antibodies to pertactin.

Pertussis is a vaccine-preventable disease, and its recent resurgence might be attributable to the emergence of strains that differ genetically from the vaccine strain. We describe a novel pertussis isolate-based surveillance system and a core genome multilocus sequence typing scheme to assess Bordetella pertussis genetic variability and investigate the increased incidence of pertussis in Austria. During 2018-2020, we obtained 123 B. pertussis isolates and typed them with the new scheme (2,983 targets and preliminary cluster threshold of

Recent reemergence of pertussis (whooping cough) in highly vaccinated populations and rapid expansion of Bordetella pertussis strains lacking pertactin (PRN), a common acellular vaccine antigen, have raised the specter of vaccine-driven evolution and potential return of what was once the major killer of children. The discovery that most circulating B. pertussis strains in the United States have acquired new and independent disruptive mutations in PRN is compelling evidence of strong selective pressure. However, the other 4 antigens included in acellular vaccines do not appear to be selected against so rapidly. We consider 3 aspects of PRN that distinguish it from other vaccine antigens, which might, individually or collectively, explain why only this antigen is being precipitously eliminated. An understanding of the increase in PRN-deficient strains should provide useful information for the current search for new protective antigens and provide broader lessons for the design of improved subunit vaccines.

Bordetella pertussis is the causative agent of whooping cough, a highly contagious respiratory disease. Pertussis toxin (PT), a major virulence factor secreted by B. pertussis, is an AB5-type protein complex topologically related to cholera toxin. The PT protein complex is internalized by host cells and follows a retrograde trafficking route to the endoplasmic reticulum, where it subsequently dissociates. The released enzymatic S1 subunit is then translocated from the endoplasmic reticulum into the cytosol and subsequently ADP-ribosylates the inhibitory alpha-subunits (Gαi) of heterotrimeric G proteins, thus promoting dysregulation of G protein-coupled receptor signaling. However, the mechanistic details of the ADP-ribosylation activity of PT are not well understood. Here, we describe crystal structures of the S1 subunit in complex with nicotinamide adenine dinucleotide (NAD+), with NAD+ hydrolysis products ADP-ribose and nicotinamide, with NAD+ analog PJ34, and with a novel NAD+ analog formed upon S1 subunit crystallization with 3-amino benzamide and NAD+, which we name benzamide amino adenine dinucleotide. These crystal structures provide unprecedented insights into pre- and post-NAD+ hydrolysis steps of the ADP-ribosyltransferase activity of PT. We propose that these data may aid in rational drug design approaches and further development of PT-specific small-molecule inhibitors.

Bordetella pertussis not expressing pertactin has increased in countries using acellular pertussis vaccines (ACV). The deficiency is mostly caused by pertactin gene disruption by IS481. To assess the effect of the transition from whole-cell vaccine to ACV on the emergence of B. pertussis not expressing pertactin in Spain, we studied 342 isolates collected during 1986-2018. We identified 93 pertactin-deficient isolates. All were detected after introduction of ACV and represented 38% of isolates collected during the ACV period; 58.1% belonged to a genetic cluster of isolates carrying the unusual prn::del(-292, 1340) mutation. Pertactin inactivation by IS481 insertion was identified in 23.7% of pertactin-deficient isolates, arising independently multiple times and in different phylogenetic branches. Our findings support the emergence and dissemination of a cluster of B. pertussis with an infrequent mechanism of pertactin disruption in Spain, probably resulting from introduction of ACV. This work was supported by the Ministerio de Economía y Competitividad, Instituto de Salud Carlos III, and cofinanced by the European Regional Development Fund “A Way to Achieve Europe” (Spanish Network for Research in Infectious Diseases, grant no. FIS PI18/00703) and by the Centro de Investigación Biomédica en Red (CIBER de Enfermedades Infecciosas), the Red Española de Investigación en Patología Infecciosa (grant no. CB21/13/00054). A.M.C. is supported by the Agència de Gestió d’Ajuts Universitaris i de Recerca de la Generalitat de Catalunya at Vall d’Hebron Institut de Recerca (Ajuts per a la Contractació de Personal Investigador FI, grant no. 2020FI_B2_00145) and by the Spanish Network for Research in Infectious Diseases (grant no. RD16/ 0016/0003). A.M.M. is supported by a grant from the Fondo de Investigación Sanitaria at Vall d’Hebron Institut de Recerca (Contratos Predoctorales de Formación en Investigación, grant no. FI19/00315). Sí

Vaccine manufacturers have recently focused on the development of in vitro potency assays to promote 3R's strategy to replace animal testing. To be able to develop an in vitro potency assay, the immunological characteristics of the monoclonal antibodies used in the assay should be well understood as these antibodies likely reflect the biological activity of a vaccine product. The PRN antigen is one of the immunogenic antigens included in many commercialized acellular pertussis vaccines. Development of an in vitro potency assay for PRN is challenging as the biological properties of PRN are not well understood. In addition, binding of Bordetella pertussis to human cells occurs through multiple bacterial molecules, which makes it very challenging to assess if antibodies contribute to prevention of bacterial adhesion. To overcome these challenges, the functionality of several in-house anti-PRN mAbs has been investigated through a novel approach using PRN-coated beads. We were able to consistently quantify the inhibition of PRN-mediated adhesion for each anti-PRN mAb. Application of the protein-coated beads model has not only enabled screening of functional anti-PRN mAbs but can also be expanded for screening of antibodies against other bacterial or viral antigens.

Since acellular vaccines (ACV) were introduced in Australia, epidemic Bordetella pertussis strains changed from single nucleotide polymorphism (SNP) cluster II to SNP cluster I. Our previous proteomic analysis identified potential proteomic adaptations in the whole cell and secretome of SNP cluster I. Additionally, current ACVs were shown to be less efficacious against cluster I in mice models and there is a pressing need to discover new antigens to improve the ACV. One important source of novel antigens is the surfaceome. Therefore, in this study we established surface shaving in B. pertussis to compare the surfaceome of SNP cluster I (L1423) and II (L1191), and identify novel surface antigens for vaccine development. Surface shaving using 1 μg of trypsin for 5 min identified 126 proteins with the most abundant being virulence-associated and known outer membrane proteins. Cell viability counts showed minimal lysis from shaving. The proportion of immunogenic proteins was higher in the surfaceome than in the whole cell and secretome. Key differences in the surfaceome were identified between SNP cluster I and II, consistent with those identified in the whole cell proteome and secretome. These differences include unique transport proteins and decreased immunogenic proteins in L1423, and provides further evidence of proteomic adaptation in SNP cluster I. Finally, a comparison of proteins in each sub-proteome identified 22 common proteins. These included 11 virulence proteins (Prn, PtxA, FhaB, CyaA, TcfA, SphB1, Vag8, BrkA, BopD, Bsp22 and BipA) and 11 housekeeping proteins (TuF, CtpA, TsF, OmpH, GltA, SucC, SucD, FusA, GroEL, BP3330 and BP3561) which were immunogenic, essential and consistently expressed thus demonstrating their potential as future targets. This study established surface shaving in B. pertussis, confirmed key expression differences and identified unknown surface proteins which may be potential vaccine antigens.

Noncoding small RNAs (sRNAs) are crucial for the posttranscriptional regulation of gene expression in all organisms and are known to be involved in the regulation of bacterial virulence. In the human pathogen Bordetella pertussis, which causes whooping cough, virulence is controlled primarily by the master two-component system BvgA (response regulator)/BvgS (sensor kinase). In this system, BvgA is phosphorylated (Bvg+ mode) or nonphosphorylated (Bvg− mode), with global transcriptional differences between the two. B. pertussis also carries the bacterial sRNA chaperone Hfq, which has previously been shown to be required for virulence. Here, we conducted transcriptomic analyses to identify possible B. pertussis sRNAs and to determine their BvgAS dependence using transcriptome sequencing (RNA-seq) and the prokaryotic sRNA prediction program ANNOgesic. We identified 143 possible candidates (25 Bvg+ mode specific and 53 Bvg− mode specific), of which 90 were previously unreported. Northern blot analyses confirmed all of the 10 ANNOgesic candidates that we tested. Homology searches demonstrated that 9 of the confirmed sRNAs are highly conserved among B. pertussis, Bordetella parapertussis, and Bordetella bronchiseptica, with one that also has homologues in other species of the Alcaligenaceae family. Using coimmunoprecipitation with a B. pertussis FLAG-tagged Hfq, we demonstrated that 3 of the sRNAs interact directly with Hfq, which is the first identification of sRNA binding to B. pertussis Hfq. Our study demonstrates that ANNOgesic is a highly useful tool for the identification of sRNAs in this system and that its combination with molecular techniques is a successful way to identify various BvgAS-dependent and Hfq-binding sRNAs. IMPORTANCE Noncoding small RNAs (sRNAs) are crucial for posttranscriptional regulation of gene expression in all organisms and are known to be involved in the regulation of bacterial virulence. We have investigated the presence of sRNAs in the obligate human pathogen B. pertussis, using transcriptome sequencing (RNA-seq) and the recently developed prokaryotic sRNA search program ANNOgesic. This analysis has identified 143 sRNA candidates (90 previously unreported). We have classified their dependence on the B. pertussis two-component system required for virulence, namely, BvgAS, based on their expression in the presence/absence of the phosphorylated response regulator BvgA, confirmed several by Northern analyses, and demonstrated that 3 bind directly to B. pertussis Hfq, the RNA chaperone involved in mediating sRNA effects. Our study demonstrates the utility of combining RNA-seq, ANNOgesic, and molecular techniques to identify various BvgAS-dependent and Hfq-binding sRNAs, which may unveil the roles of sRNAs in pertussis pathogenesis.

Pulmonary infections caused by Bordetella pertussis used to be the prime cause of infant mortality in the pre-vaccine era and mouse models of pertussis pneumonia served in characterization of B. pertussis virulence mechanisms. However, the biologically most relevant catarrhal disease stage and B. pertussis transmission has not been adequately reproduced in adult mice due to limited proliferation of the human-adapted pathogen on murine nasopharyngeal mucosa. We used immunodeficient C57BL/6J MyD88 KO mice to achieve B. pertussis proliferation to human-like high counts of 108 viable bacteria per nasal cavity to elicit rhinosinusitis accompanied by robust shedding and transmission of B. pertussis bacteria to adult co-housed MyD88 KO mice. Experiments with a comprehensive set of B. pertussis mutants revealed that pertussis toxin, adenylate cyclase toxin-hemolysin, the T3SS effector BteA/BopC and several other known virulence factors were dispensable for nasal cavity infection and B. pertussis transmission in the immunocompromised MyD88 KO mice. In contrast, mutants lacking the filamentous hemagglutinin (FhaB) or fimbriae (Fim) adhesins infected the nasal cavity poorly, shed at low levels and failed to productively infect co-housed MyD88 KO or C57BL/6J mice. FhaB and fimbriae thus appear to play a critical role in B. pertussis transmission. The here-described novel murine model of B. pertussis-induced nasal catarrh opens the way to genetic dissection of host mechanisms involved in B. pertussis shedding and to validation of key bacterial transmission factors that ought to be targeted by future pertussis vaccines.

Background Bordetella pertussis is the main agent of whooping cough. Vaccination with acellular pertussis vaccines has been largely implemented in high-income countries. These vaccines contain 1 to 5 antigens: pertussis toxin (PT), filamentous haemagglutinin (FHA), pertactin (PRN) and/or fimbrial proteins (FIM2 and FIM3). Monitoring the emergence of B. pertussis isolates that might partially escape vaccine-induced immunity is an essential component of public health strategies to control whooping cough. Aim We aimed to investigate temporal trends of fimbriae serotypes and vaccine antigen-expression in B. pertussis over a 23-year period in France (1996–2018). Methods Isolates (n = 2,280) were collected through hospital surveillance, capturing one third of hospitalised paediatric pertussis cases. We assayed PT, FHA and PRN production by Western blot (n = 1,428) and fimbriae production by serotyping (n = 1,058). Molecular events underlying antigen deficiency were investigated by genomic sequencing. Results The proportion of PRN-deficient B. pertussis isolates has increased steadily from 0% (0/38) in 2003 to 48.4% (31/64) in 2018 (chi-squared test for trend, p 481 insertion within the prn gene or a 22 kb genomic inversion involving the prn promoter sequence, indicative of convergent evolution. FIM2-expressing isolates have emerged since 2011 at the expense of FIM3. Conclusions B. pertussis is evolving through the rapid increase of PRN-deficient isolates and a recent shift from FIM3 to FIM2 expression. Excluding PRN, the loss of vaccine antigen expression by circulating B. pertussis isolates is epidemiologically insignificant.

RTX leukotoxins are a diverse family of prokaryotic virulence factors that are secreted by the type 1 secretion system (T1SS) and target leukocytes to subvert host defenses. T1SS substrates all contain a C-terminal RTX domain that mediates recruitment to the T1SS and drives secretion via a Brownian ratchet mechanism. Neutralizing antibodies against the Bordetella pertussis adenylate cyclase toxin, an RTX leukotoxin essential for B. pertussis colonization, have been shown to target the RTX domain and prevent binding to the αMβ2 integrin receptor. Knowledge of the mechanisms by which antibodies bind and neutralize RTX leukotoxins is required to inform structure-based design of bacterial vaccines, however, no structural data are available for antibody binding to any T1SS substrate. Here, we determine the crystal structure of an engineered RTX domain fragment containing the αMβ2-binding site bound to two neutralizing antibodies. Notably, the receptor-blocking antibodies bind to the linker regions of RTX blocks I–III, suggesting they are key neutralization-sensitive sites within the RTX domain and are likely involved in binding the αMβ2 receptor. As the engineered RTX fragment contained these key epitopes, we assessed its immunogenicity in mice and showed that it elicits similar neutralizing antibody titers to the full RTX domain. The results from these studies will support the development of bacterial vaccines targeting RTX leukotoxins, as well as next-generation B. pertussis vaccines., Author summary Diverse bacterial pathogens use the type 1 secretion system (T1SS) to secrete RTX leukotoxins, which target host leukocytes during infection. T1SS substrates all contain a repetitive C-terminal ‘RTX’ domain that adopts a characteristic β-roll fold and is involved in secretion. Notably, The RTX domain of Bordetella pertussis adenylate cyclase toxin (ACT) mediates leukocyte targeting via binding to the αMβ2 integrin receptor, and antibodies that block receptor binding neutralize toxin activity. However, ACT also contains multiple non-neutralizing epitopes, and precise knowledge of the sites targeted by neutralizing antibodies is desirable for vaccine design. Here we determine the crystal structure of an ACT fragment in complex with two neutralizing antibodies and define the key neutralization-sensitive sites within the RTX domain. This first structure of a heterotypic protein–protein interaction formed by an RTX domain suggests the linker regions between β-roll segments engage binding partners.

Infection by the bacterium Bordetella pertussis continues to cause considerable morbidity and mortality worldwide. Many current acellular pertussis vaccines include the antigen pertactin, which has presumptive adhesive and immunomodulatory activities, but is rapidly lost from clinical isolates after the introduction of these vaccines. To better understand the contributions of pertactin antibodies to protection and pertactin's role in pathogenesis, we isolated and characterized recombinant antibodies binding four distinct epitopes on pertactin. We demonstrate that four of these antibodies bind epitopes that are conserved across all three classical Bordetella strains, and competition assays further showed that antibodies binding these epitopes are also elicited by B. pertussis infection of baboons. Surprisingly, we found that representative antibodies binding each epitope protected mice against experimental B. pertussis infection. A cocktail of antibodies from each epitope group protected mice against a subsequent lethal dose of B. pertussis and greatly reduced lung colonization levels after sublethal challenge. Each antibody reduced B. pertussis lung colonization levels up to 100-fold when administered individually, which was significantly reduced when antibody effector functions were impaired, with no antibody mediating antibody-dependent complement-induced lysis. These data suggest that antibodies binding multiple pertactin epitopes protect primarily by the same bactericidal mechanism, which overshadows contributions from blockade of other pertactin functions. These antibodies expand the available tools to further dissect pertactin's role in infection and understand the impact of antipertactin antibodies on bacterial fitness.

Production and secretion of pertussis toxin (PT) is essential for the virulence of Bordetella pertussis. Due to the large oligomeric structure of PT, transport of the toxin across bacterial membrane barriers represents a significant hurdle that the bacteria must overcome in order to maintain pathogenicity. During the secretion process, PT undergoes a two-step transport process. The first step involves transport of the individual polypeptide chains of PT across the inner membrane utilizing a generalized secretion pathway, most likely the bacterial Sec system. The second step involves the use of a specialized apparatus to transport the toxin across the outer membrane of the bacterial cell. This apparatus, which has been termed the Ptl transporter and which is unique to the PT secretion pathway, is a member of the type IV family of bacterial transporters. Here, the current understanding of the PT secretion process is reviewed including a description of the Ptl proteins that assemble to form the transporter, the general structure of type IV transporters, the known similarities and differences between canonical type IV substrate transport and Ptl-mediated transport of PT, as well as the known sequence of events in the assembly and secretion of PT.

Whooping cough is resurging in the United States despite high vaccine coverage. The rapid rise of Bordetella pertussis isolates lacking pertactin (PRN), a key vaccine antigen, has led to concerns about vaccine-driven evolution. Previous studies showed that pertactin can mediate binding to mammalian cells in vitro and act as an immunomodulatory factor in resisting neutrophil-mediated clearance. To further investigate the role of PRN in vivo, we examined the functions of pertactin in the context of a more naturally low dose inoculation experimental system using C3H/HeJ mice that is more sensitive to effects on colonization, growth and spread within the respiratory tract, as well as an experimental approach to measure shedding and transmission between hosts. A B. bronchiseptica pertactin deletion mutant was found to behave similarly to its wild-type (WT) parental strain in colonization of the nasal cavity, trachea, and lungs of mice. However, the pertactin-deficient strain was shed from the nares of mice in much lower numbers, resulting in a significantly lower rate of transmission between hosts. Histological examination of respiratory epithelia revealed that pertactin-deficient bacteria induced substantially less inflammation and mucus accumulation than the WT strain and in vitro assays verified the effect of PRN on the induction of TNF-α by murine macrophages. Interestingly, only WT B. bronchiseptica could be recovered from the spleen of infected mice and were further observed to be intracellular among isolated splenocytes, indicating that pertactin contributes to systemic dissemination involving intracellular survival. These results suggest that pertactin can mediate interactions with immune cells and augments inflammation that contributes to bacterial shedding and transmission between hosts. Understanding the relative contributions of various factors to inflammation, mucus production, shedding and transmission will guide novel strategies to interfere with the reemergence of pertussis., Author summary B. pertussis strains lacking pertactin have been rising in prevalence especially in countries using acellular vaccines containing pertactin as a key, membrane-associated surface antigen. Previous in vivo studies revealed immunomodulatory properties of pertactin in conventional B. pertussis infection models in which roughly one million bacteria are delivered into lungs, leading to severe pneumonic disease and a strong immune response. However, natural infections begin in the nasopharyngeal region, progress slowly during a prolonged catarrhal stage, only later reaching the trachea and rarely involve the lungs. In this study, a more natural experimental system takes advantage of the ability of B. bronchiseptica, a closely related species, to naturally colonize mice with inocula as low as 5 colony forming units (CFU). In this system B. bronchiseptica can be observed to efficiently colonize, grow, spread within the respiratory tract, is shed from the nares, and transmits between hosts, allowing each of these steps to be measured and studied. Under these conditions, an isogenic pertactin deletion strain was indistinguishable from its parental strain in its abilities to colonize, grow in numbers and spread within the respiratory tract. However, the pertactin-deficient mutant was shed from these mice in lower numbers than wild type, and was defective in transmission between mice. These assays reveal novel roles of pertactin in the induction of inflammation, mucus production, shedding and transmission.

Peptidoglycan recognition proteins (PGRPs) are ubiquitous among animals and play pivotal functions in insect immunity. Non-catalytic PGRPs are involved in the activation of immune pathways by binding to the peptidoglycan (PGN), whereas amidase PGRPs are capable of cleaving the PGN into non-immunogenic compounds. Drosophila PGRP-LB belongs to the amidase PGRPs and downregulates the immune deficiency (IMD) pathway by cleaving meso-2,6-diaminopimelic (meso-DAP or DAP)-type PGN. While the recognition process is well analyzed for the non-catalytic PGRPs, little is known about the enzymatic mechanism for the amidase PGRPs, despite their essential function in immune homeostasis. Here, we analyzed the specific activity of different isoforms of Drosophila PGRP-LB towards various PGN substrates to understand their specificity and role in Drosophila immunity. We show that these isoforms have similar activity towards the different compounds. To analyze the mechanism of the amidase activity, we performed site directed mutagenesis and solved the X-ray structures of wild-type Drosophila PGRP-LB and its mutants, with one of these structures presenting a protein complexed with the tracheal cytotoxin (TCT), a muropeptide derived from the PGN. Only the Y78F mutation abolished the PGN cleavage while other mutations reduced the activity solely. Together, our findings suggest the dynamic role of the residue Y78 in the amidase mechanism by nucleophilic attack through a water molecule to the carbonyl group of the amide function destabilized by Zn2+.

The structure of a 10-kDa protein complex is one of the smallest to be determined using cryo-electron microscopy at high resolution. The structure reveals that C1-INH is sequestered in an inactivated state by burial of the reactive center loop in Vag8., Complement, contact activation, coagulation, and fibrinolysis are serum protein cascades that need strict regulation to maintain human health. Serum glycoprotein, a C1 inhibitor (C1-INH), is a key regulator (inhibitor) of serine proteases of all the above-mentioned pathways. Recently, an autotransporter protein, virulence-associated gene 8 (Vag8), produced by the whooping cough pathogen, Bordetella pertussis, was shown to bind to C1-INH and interfere with its function. Here, we present the structure of the Vag8–C1-INH complex determined using cryo-electron microscopy at a 3.6-Å resolution. The structure shows a unique mechanism of C1-INH inhibition not employed by other pathogens, where Vag8 sequesters the reactive center loop of C1-INH, preventing its interaction with the target proteases.

Current acellular-pertussis (aP) vaccines appear inadequate for long-term pertussis control because of short-lived efficacy and the increasing prevalence of pertactin-negative isolates which may negatively impact vaccine efficacy. In this study, we added fimbriae (FIM)2 and FIM3 protein to licensed 2-, 3- or 5-component aP vaccines (Pentavac(®), Boostrix(®), Adacel(®), respectively) to assess whether an aP vaccine with enhanced FIM content demonstrates enhanced efficacy. Vaccine-induced protection was assessed in an intranasal mouse challenge model. In addition, potential reactogenicity was measured by biomarkers in a human whole blood assay (WBA) in vitro and benchmarked the responses against licensed whole cell pertussis (wP) and aP vaccines including Easyfive(®), Pentavac(®) and Pentacel(®). The results show that commercial vaccines demonstrated reduced efficacy against pertactin-negative versus pertactin-positive strains. However, addition of higher amounts of FIM2/3 to aP vaccines reduced lung colonization and increased vaccine efficacy against a pertactin-negative strain in a dose- dependent manner. Improvements in efficacy were similar for FIM2 and FIM3-expressing strains. Increasing the amount of FIM2/3 proteins in aP formulations did not alter vaccine- induced biomarkers of potential reactogenicity including prostaglandin E(2), cytokines and chemokines in human newborn cord and adult peripheral blood tested in vitro. These results suggest that increasing the quantity of FIM proteins in current pertussis vaccine formulations may further enhance vaccine efficacy against B. pertussis infection without increasing the reactogenicity of the vaccine.

Background/Purpose: Although assessing the immunogenicity and protective efficacy of acellular pertussis (aP) vaccines via murine model studies faces limitations, preliminary assessments have been achieved by evaluating respiratory challenge and humoral and cellular immunity. Methods: We performed a long-term intranasal respiratory challenge with reference and clinically isolated strains of Bordetella pertussis. Simultaneously, we assessed humoral and cellular immunity for evaluating the immunogenicity of a newly developed tri-component diphtheria-tetanus-aP (DTaP) vaccine. Moreover, comparative assessment was made by performing the same evaluations with a commercially available tri-component DTaP vaccine as the positive control. Results: Both groups showed significantly increased levels of antibodies against pertussis toxin, filamentous hemagglutinin and pertactin, and the levels of interferon-γ and interleukin-10 were significantly increased after two doses of vaccination. Furthermore, since cross cell-mediated immune reactivity between the two vaccines was detected, the possibility of interchangeability was indirectly suggested. Although the positive control group showed significantly higher titers in antibody responses for filamentous hemagglutinin and pertactin compared to the experimental group, anti-pertussis toxin antibody titers of the two groups were not significantly different and the protective efficacy against the clinical and reference strains was maintained in both groups for 18 weeks. Conclusion: The results showed inferior immunogenicity of the new DTaP vaccine compared to a commercial vaccine despite comparable cellular immunity and protective efficacy. Some efforts are necessary for improving immunogenicity against filamentous hemagglutinin and pertactin before conducting human clinical trials. Keywords: Diphtheria-tetanus-acellular pertussis vaccine, Immunogenicity, Efficacy, Mice

Bordetella pertussis is a highly contagious pathogen which causes whooping cough in humans. A major pathophysiology of infection is the extrusion of ciliated cells and subsequent disruption of the respiratory mucosa. Tracheal cytotoxin (TCT) is the only virulence factor produced by B. pertussis that has been able to recapitulate this pathology in animal models. This pathophysiology is well characterized in a hamster tracheal model, but human data are lacking due to scarcity of donor material. We assessed the impact of TCT and lipopolysaccharide (LPS) on the functional integrity of the human airway mucosa by using in vitro airway mucosa models developed by co-culturing human tracheobronchial epithelial cells and human tracheobronchial fibroblasts on porcine small intestinal submucosa scaffold under airlift conditions. TCT and LPS either alone and in combination induced blebbing and necrosis of the ciliated epithelia. TCT and LPS induced loss of ciliated epithelial cells and hyper-mucus production which interfered with mucociliary clearance. In addition, the toxins had a disruptive effect on the tight junction organization, significantly reduced transepithelial electrical resistance and increased FITC-Dextran permeability after toxin incubation. In summary, the results indicate that TCT collaborates with LPS to induce the disruption of the human airway mucosa as reported for the hamster tracheal model.

Bordetella pertussis causes the severe childhood disease whooping cough, by releasing several toxins, including pertussis toxin (PT) as a major virulence factor. PT is an AB5-type toxin, and consists of the enzymatic A-subunit PTS1 and five B-subunits, which facilitate binding to cells and transport of PTS1 into the cytosol. PTS1 ADP-ribosylates ��-subunits of inhibitory G-proteins (G��i) in the cytosol, which leads to disturbed cAMP signaling. Since PT is crucial for causing severe courses of disease, our aim is to identify new inhibitors against PT, to provide starting points for novel therapeutic approaches. Here, we investigated the effect of human antimicrobial peptides of the defensin family on PT. We demonstrated that PTS1 enzyme activity in vitro was inhibited by ��-defensin-1 and -5, but not ��-defensin-1. The amount of ADP-ribosylated G��i was significantly reduced in PT-treated cells, in the presence of ��-defensin-1 and -5. Moreover, both ��-defensins decreased PT-mediated effects on cAMP signaling in the living cell-based interference in the G��i-mediated signal transduction (iGIST) assay. Taken together, we identified the human peptides ��-defensin-1 and -5 as inhibitors of PT activity, suggesting that these human peptides bear potential for developing novel therapeutic strategies against whooping cough., publishedVersion

Respiratory infections caused by Bordetella pertussis are reemerging despite high pertussis vaccination coverage. Since the introduction of the acellular pertussis vaccine in the late twentieth century, circulating B. pertussis strains increasingly lack expression of the vaccine component pertactin (Prn). In some countries, up to 90% of the circulating B. pertussis strains are deficient in Prn. To better understand the resurgence of pertussis, we investigated the response of human monocyte-derived dendritic cells (moDCs) to naturally circulating Prn-expressing (Prn-Pos) and Prn-deficient (Prn-Neg) B. pertussis strains from 2016 in the Netherlands. Transcriptome analysis of moDC showed enriched IFNα response-associated gene expression after exposure to Prn-Pos B. pertussis strains, whereas the Prn-Neg strains induced enriched expression of interleukin- and TNF-signaling genes, as well as other genes involved in immune activation. Multiplex immune assays confirmed enhanced proinflammatory cytokine secretion by Prn-Neg stimulated moDC. Comparison of the proteomes from the Prn-Pos and Prn-Neg strains revealed, next to the difference in Prn, differential expression of a number of other proteins including several proteins involved in metabolic processes. Our findings indicate that Prn-deficient B. pertussis strains induce a distinct and stronger immune activation of moDCs than the Prn-Pos strains. These findings highlight the role of pathogen adaptation in the resurgence of pertussis as well as the effects that vaccine pressure can have on a bacterial population.

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.

Pertussis is a highly contagious disease for which prompt, point-of-care diagnosis remains an unmet clinical need. Results from conventional test modalities (nucleic acid detection, serology, and culture) take hours to days. To overcome this challenge, we identified a new biomarker (tracheal colonization factor A, TcfA) for detection of Bordetella pertussis infection by lateral flow immunoassay (LFIA). We developed a library of 28 epitope-mapped monoclonal antibodies against TcfA and incorporated three antibodies into a LFIA. The LFIA did not cross-react with common bacterial or fungal organisms, but did react with nine distinct B. pertussis strains. The minimal linear epitope sequences targeted by the LFIA were conserved in 98% of 954 B. pertussis isolates collected across 12 countries from 1949–2017. The LFIA’s limit of detection was 3.0 × 105 CFU/mL with B. pertussis cells in buffer, 6.2 × 105 CFU/mL with nasopharyngeal washes from a non-human primate model, and 2.3 ng/mL with recombinant TcfA. The LFIA reacted with patient nasopharyngeal swab specimens containing as few as 1.8 × 106B. pertussis genomes/mL and showed no false-positives. Rapid (B. pertussis infection is feasible and may facilitate early detection of pertussis.

The pertussis vaccination is highly recommended for infants, children, and pregnant women. Despite a high coverage of vaccination, pertussis continues to be of public health concern as a re-emerging infectious disease. The mechanism by which vaccine-elicited anti-pertussis antibodies mediate direct bactericidal effects is poorly understood. In this study, we showed that the interaction ofB. pertussiswith A549 epithelial cells induce release of biological factors which enhance bacteria growth. Complement-depleted antisera from vaccine-immunized guinea pigs or monoclonal antibodies targeting FHA and FIM mediate bacteria aggregation and elicit bactericidal effects. Ourin vitroresults indicated that aggregation of bacteria through anti-FIM and anti-FHA specific antibodies is one of the major biological mechanisms to clear bacterial infections and restore epithelial cell survivalin vitro. Our data also indicates that the anti-pertussis antibodies reduce secretion of proinflammatory chemokines and cytokines by preventing interaction ofB. pertussiswith host cells. The results of this study not only demonstrate mechanism of action of anti-FIM and anti-FHA antibodies, but also opens translational applications for potential therapeutic approaches or development of analytical assays such asin vitropotency assays.

Much attention is being paid to conformational biases in the ensembles of intrinsically disordered proteins. However, it is currently unknown whether or how conformational biases within the disordered ensembles of foldable proteins affect function in vivo. Recently, we demonstrated that water can be a good solvent for unfolded polypeptide chains, even those with a hydrophobic and charged sequence composition typical of folded proteins. These results run counter to the generally accepted model that protein folding begins with hydrophobicity-driven chain collapse. Here we investigate what other features, beyond amino acid composition, govern chain collapse. We found that local clustering of hydrophobic and/or charged residues leads to significant collapse of the unfolded ensemble of pertactin, a secreted autotransporter virulence protein from Bordetella pertussis, as measured by small angle X-ray scattering (SAXS). Sequence patterns that lead to collapse also correlate with increased intermolecular polypeptide chain association and aggregation. Crucially, sequence patterns that support an expanded conformational ensemble enhance pertactin secretion to the bacterial cell surface. Similar sequence pattern features are enriched across the large and diverse family of autotransporter virulence proteins, suggesting sequence patterns that favor an expanded conformational ensemble are under selection for efficient autotransporter protein secretion, a necessary prerequisite for virulence. More broadly, we found that sequence patterns that lead to more expanded conformational ensembles are enriched across water-soluble proteins in general, suggesting protein sequences are under selection to regulate collapse and minimize protein aggregation, in addition to their roles in stabilizing folded protein structures.

Pathogenic Bordetella bacteria release a neurotropic dermonecrotic toxin (DNT) that is endocytosed into animal cells and permanently activates the Rho family GTPases by polyamination or deamidation of the glutamine residues in their switch II regions (e.g., Gln63 of RhoA). DNT was found to enable high level colonization of the nasal cavity of pigs by B. bronchiseptica and the capacity of DNT to inhibit differentiation of nasal turbinate bone osteoblasts causes atrophic rhinitis in infected pigs. However, it remains unknown whether DNT plays any role also in virulence of the human pathogen B. pertussis and in pathogenesis of the whooping cough disease. We report a procedure for purification of large amounts of LPS-free recombinant DNT that exhibits a high biological activity on cells expressing the DNT receptors Cav3.1 and Cav3.2. Electron microscopy and single particle image analysis of negatively stained preparations revealed that the DNT molecule adopts a V-shaped structure with well-resolved protein domains. These results open the way to structure&ndash, function studies on DNT and its interactions with airway epithelial layers.

Background Diarrhoeagenic Escherichia coli (DEC) infections are common in children in low-middle income countries (LMICs). However, detecting the various DEC pathotypes is complex as they cannot be differentiated by classical microbiology. We developed four multiplex real-time PCR assays were to detect virulence markers of six DEC pathotypes; specificity was tested using DEC controls and other enteric pathogens. PCR amplicons from the six E. coli pathotypes were purified and amplified to be used to optimize PCR reactions and to calculate reproducibility. After validation, these assays were applied to clinical samples from healthy and diarrhoeal Vietnamese children and associated with clinical data. Results The multiplex real-time PCRs were found to be reproducible, and specific. At least one DEC variant was detected in 34.7% (978/2815) of the faecal samples from diarrhoeal children; EAEC, EIEC and atypical EPEC were most frequent Notably, 41.2% (205/498) of samples from non-diarrhoeal children was positive with a DEC pathotype. In this population, only EIEC, which was detected in 34.3% (99/289) of diarrhoeal samples vs. 0.8% (4/498) non-diarrhoeal samples (p Conclusions This approach revealed high carriage rates of DEC pathotypes among Vietnamese children. We describe a novel diagnostic approach for DEC, which provides baseline data for future surveillance studies assessing DEC burden in LMICs.

We performed RNA sequencing on Bordetella pertussis, the causative agent of whooping cough, and identified nine novel small RNAs (sRNAs) that were transcribed during the bacterial colonization of murine tracheas. Among them, four sRNAs were more strongly expressed in vivo than in vitro. Moreover, the expression of eight sRNAs was not regulated by the BvgAS two‐component system, which is the master regulator for the expression of genes contributing to the bacterial infection. The present results suggest a BvgAS‐independent gene regulatory system involving the sRNAs that is active during B. pertussis infection.

International audience; Pneumococcal Surface Protein A (PspA) has been successfully tested as vaccine candidate against Streptococcus pneumoniae infections. Vaccines able to induce PspA-specific antibodies and Th1 cytokines usually provide protection in mice. We have shown that the whole cell pertussis vaccine (wP) or components from acellular pertussis vaccines, such as Pertussis Toxin or Filamentous Hemagglutinin (FHA), are good adjuvants to PspA, suggesting that combined pertussis-PspA vaccines would be interesting strategies against the two infections. Here, we evaluated the potential of wP as a delivery vector to PspA. Bordetella pertussis strains producing a PspA from clade 4 (PspA4Pro) fused to the N-terminal region of FHA (Fha44) were constructed and inactivated with formaldehyde for the production of wPPspA4Pro. Subcutaneous immunization of mice with wPPspA4Pro induced low levels of anti-PspA4 IgG, even after 3 doses, and did not protect against a lethal pneumococcal challenge. Prime-boost strategies using wPPspA4Pro and PspA4Pro showed that there was no advantage in using the wPPspA4Pro vaccine. Immunization of mice with purified PspA4Pro induced higher levels of antibodies and protection against pneumococcal infection than the prime-boost strategies. Finally, purified Fha44:PspA4Pro induced high levels of anti-PspA4Pro IgG, but no protection, suggesting that the antibodies induced by the fusion protein were not directed to protective epitopes.

Contains fulltext : 225116.pdf (Publisher’s version ) (Open Access) 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.

Determination of protein concentration in vaccines containing aluminum salt adjuvant typically necessitates desorption of the protein prior to analysis. Here we describe a method based on the intrinsic fluorescence of tyrosine and tryptophan that requires no desorption of proteins. Adjuvanted formulations of three model Bordetella pertussis antigens were excited at 280 nm and their emission spectra collected from 290 to 400 nm. Emission spectra of protein antigens in the presence of aluminum salt adjuvants were able to be detected, the effects of adjuvants on the spectra were analyzed, and linear regressions were calculated. The fluorescence method proved to be very sensitive with a limit of quantification between 0.4 and 4.4 µg/mL and limit of linearity between 100 and 200 µg/mL, across the formulations tested. The fluorescence method was found to be influenced by adjuvant presence, type of adjuvant, adjuvant concentration, buffer and pH conditions. The method also demonstrated ability to monitor the percent adsorption of antigens to the adjuvants. Furthermore, intrinsic fluorescence showed good correlation with micro-Kjeldahl elemental assay in quantifying protein concentration. Being a non-invasive, quick and sensitive method, intrinsic fluorescence has the potential to be utilized as a high throughput tool for vaccine development and conceivably implemented in-line, using in-line fluorimeters, to monitor antigen concentration during formulation processing.

Bordetella pertussis (B. pertussis) infection occasionally causes severe respiratory infections in children. Potential association between virulence-associated gene alleles and severe clinical outcomes has been suggested; however, frequencies of these alleles in pediatric patients with severe pertussis have not been clarified. We retrospectively tested stored respiratory samples collected from B. pertussis-positive patients by polymerase chain reaction targeting for major virulence-associated genes; fimbrae (fim) serotype 3 (fim3), pertactin (prn), pertussis toxin A (ptxA), and pertussis toxin promotor (ptxP). Based on the identified sequences, multilocus sequence typing (MLST) was conducted. Association of gene allele frequency and clinical outcomes such as management in pediatric intensive care unit, intubation, and mortality was analyzed. Out of 25 patients with available samples, the most prevalent allele for each virulence-associated gene was fim3A (17/21, 83%), ptxA1 (20/23, 87%), prn2 (13/16, 81%), and ptxP3 (14/17, 82%). In the study, total of 3 MLST types were identified; MLST-1 from 3 patients (19%), MLST-2 from 10 patients (63%), and MLST-4 from 3 patients (19%). Detection rate of the major MLST type; MLST-2 was significantly higher for patients who required intubation compared to those who did not (6/6, 100% vs 4/10, 40%; p = 0.034). In conclusion, MLST-2 was the most common MLST type in B. pertussis isolated from Japanese children with severe pertussis. Further studies investigating its causal association with disease severity is warranted.

Among the factors associated with the resurgence of whooping cough, special emphasis has been given to pathogen adaptation after the introduction of the acellular vaccine (ACV). To assess the impact of the vaccine transition strategy from whole-cell vaccine (WCV) to ACV on population dynamics of Bordetella pertussis in Barcelona (Spain), we studied 339 isolates collected from 1986 to 2015 by PFGE and multi-locus variable-number tandem repeat analysis (MLVA). Additionally, allelic variants for the pertussis toxin and its promoter, pertactin, type 3 fimbriae and fimbrial serotyping were assessed to determine its antigenic drift. A shift was observed in the B. pertussis population as well as in its antigenic profile concurrently with the introduction of ACV in Barcelona. Four out of the five most prevalent PFGE profiles were replaced by new profiles following the ACV introduction. MLVA type 27 was the dominant genotype, and its frequency increased from 25% to 79.3% after WCV replacement. Antigen typing demonstrated the emergence of prn2, ptxP3, fim3-2 and a shift from the fimbriae 3 to the fimbriae 2 serotypes after the ACV introduction. Our findings support the presence of population and antigenic dynamic changes in B. pertussis likely driven by the introduction of ACV.

Pertussis resurgence had been attributed to waning vaccine immunity and Bordetella pertussis adaptation to escape vaccine-induced immunity. Circulating bacteria differ genotypically from strains used in production of pertussis vaccine. Pertactin-deficient strains are highly prevalent in countries that use acellular vaccine (aP), suggesting strong aP-imposed selection of circulating bacteria. To corroborate this hypothesis, systematic studies on pertactin prevalence of infection in countries using whole-cell vaccine are needed. We provide pertussis epidemiologic data and molecular characterization of B. pertussis isolates from Buenos Aires, Argentina, during 2000-2017. This area used primary vaccination with whole-cell vaccine. Since 2002, pertussis case incidences increased at regular 4-year outbreaks; most cases were in infants

We constructed a food-grade expression system harboring a F1S1 fusion protein of Bordetella pertussis to be produced in Lactococcus lactis NZ3900 as a new oral vaccine model against whooping cough, caused by B. pertussis. F1S1 was composed of N-terminally truncated S1 subunit of pertussis toxin and type I immunodominant domain of filamentous hemagglutinin which are both known as protective immunogens against pertussis. The recombinant L. lactis was administered via oral or intranasal routes to BALB/c mice and the related specific systemic and mucosal immune responses were then evaluated. The results indicated significantly higher levels of specific IgA in the lung extracts and IgG in sera of mucosally-immunized mice, compared to their controls. It was revealed that higher levels of IgG2a, compared to IgG1, were produced in all mucosally-immunized mice. Moreover, immunized mice developed Th1 responses with high levels of IFN-γ production by the spleen cells. These findings provide evidence for L. lactis to be used as a suitable vehicle for expression and delivery of F1S1 fusion protein to mucosa and induction of appropriate systemic and mucosal immune responses against pertussis., Highlights • Lactococcus lactis was used for expression of fusion protein from Bordetella pertussis. • BALB/c mice were immunized via oral or intranasal routes with recombinant L. lactis. • Strong mucosal and Th1 systemic immune responses were developed. • L. lactis is a suitable vehicle for expression and delivery of B. pertussis fusion protein.

Since acellular vaccines (ACV) were introduced in Australia, epidemic Bordetella pertussis strains changed from single nucleotide polymorphism (SNP) cluster II to SNP cluster I. Our previous proteomic analysis identified potential proteomic adaptations in the whole cell and secretome of SNP cluster I. Additionally, current ACVs were shown to be less efficacious against cluster I in mice models and there is a pressing need to discover new antigens to improve the ACV. One important source of novel antigens is the surfaceome. Therefore, in this study we established surface shaving in B. pertussis to compare the surfaceome of SNP cluster I (L1423) and II (L1191), and identify novel surface antigens for vaccine development. Surface shaving using 1 μg of trypsin for 5 min identified 126 proteins with the most abundant being virulence-associated and known outer membrane proteins. Cell viability counts showed minimal lysis from shaving. The proportion of immunogenic proteins was higher in the surfaceome than in the whole cell and secretome. Key differences in the surfaceome were identified between SNP cluster I and II, consistent with those identified in the whole cell proteome and secretome. These differences include unique transport proteins and decreased immunogenic proteins in L1423, and provides further evidence of proteomic adaptation in SNP cluster I. Finally, a comparison of proteins in each sub-proteome identified 22 common proteins. These included 11 virulence proteins (Prn, PtxA, FhaB, CyaA, TcfA, SphB1, Vag8, BrkA, BopD, Bsp22 and BipA) and 11 housekeeping proteins (TuF, CtpA, TsF, OmpH, GltA, SucC, SucD, FusA, GroEL, BP3330 and BP3561) which were immunogenic, essential and consistently expressed thus demonstrating their potential as future targets. This study established surface shaving in B. pertussis, confirmed key expression differences and identified unknown surface proteins which may be potential vaccine antigens.

Serological surveillance of pertussis antibodies was performed in 118 children aged 1-12 years. The positivity of pertussis toxin (PT) antibodies was low at 4-6 years and significantly higher at 8-9 years, compared with those at 6 years. Fimbriae 2 (Fim2) antibody showed similar response to the PT antibody. Higher antibody titers against Fim3 were observed among subjects ≥5 years and highest at 8 years. Data demonstrated that the vaccine-induced antibodies decayed by 4-5 years and subclinical pertussis infection was suspected thereafter, suggesting the need for additional dose at around 4-5 years.

The antigens for acellular pertussis vaccines are made up of protein components that are purified directly from Bordetella pertussis (B. pertussis) bacterial fermentation. As such, there are additional B. pertussis toxins that must be monitored as residuals during process optimization. This paper describes a liquid chromatography mass spectrometry (LC-MS) method for simultaneous analysis of residual protein toxins adenylate cyclase toxin (ACT) and dermonecrotic toxin (DNT), as well as a small molecule glycopeptide, tracheal cytotoxin (TCT) in a Pertussis toxin vaccine antigen. A targeted LC-MS technique called multiple reaction monitoring (MRM) is used for quantitation of ACT and TCT, which have established limits in drug product formulations. However, DNT is currently monitored in an animal test, which does not have an established quantitative threshold. New approaches for DNT testing are discussed, including a novel standard based on concatenated quantitation sequences for ACT and DNT. Collectively, the method represents a “3-in-1” analytical simplification for monitoring process-related residuals during development of B. pertussis vaccines.

An expanded view of disordered proteins Disordered proteins sample an ensemble of conformations, but it has remained unclear how compact these conformations are in water. Polymer physics relates the radius of gyration ( R g ) to solvent quality, with more chain collapse occurring in poorer solvents. Riback et al. developed an analysis scheme that allows them to extract solvent quality and R g from a single small-angle x-ray scattering measurement. Applying this method, they found that even disordered proteins with low net charge and high hydrophobicity remain expanded in water. Science , this issue p. 238