Optimal Detection of Latent Mycobacterium tuberculosis Infection by Combined Heparin-Binding Hemagglutinin (HBHA) and Early Secreted Antigenic Target 6 (ESAT-6) Whole-Blood Interferon Gamma Release Assays.

Optimal detection of latent tuberculosis (TB) infection (LTBI) remains a challenge, although it is essential to reach the goal of TB elimination. Our objective was to develop and clinically evaluate a user-friendly, 24-h, whole-blood (WB) interferon gamma (IFN-γ) release assay (IGRA) improving the detection of LTBI, compared to available tests. One milliliter of blood was divided into four aliquots and in vitro stimulated for 24 h with two different stage-specific mycobacterial antigens, i.e., heparin-binding hemagglutinin (HBHA) and early secreted antigenic target 6 (ESAT-6), a latency-associated antigen and a bacterial replication-related antigen, respectively, in addition to positive and negative controls. Clinical evaluation was performed on two independent cohorts of carefully selected subjects, i.e., a training cohort of 83 individuals and a validation cohort of 69 individuals. Both cohorts comprised LTBI subjects (asymptomatic people with a positive tuberculin skin test result and potential exposure to TB index cases), patients with active TB (aTB), and noninfected controls. The sensitivity and specificity of the WB-HBHA-IGRA to identify LTBI subjects among asymptomatic individuals were 93%. Combining the results in response to HBHA and ESAT-6 allowed us to identify LTBI subgroups. One group, with IFN-γ responses to HBHA only, was easily differentiated from patients with aTB. The other group, responding to both antigens like the aTB group, is likely at risk to reactivate the infection and should be prioritized for prophylactic anti-TB treatment. The combined WB-IGRA may be offered to clinicians for the selection of LTBI subjects to benefit from prophylactic treatment.

Attenuated Bordetella pertussis BPZE1 protects against allergic airway inflammation and contact dermatitis in mouse models.

BACKGROUND: We previously reported that prior nasal administration of highly attenuated Bordetella pertussis BPZE1 provides effective and sustained protection against lethal challenge with influenza A viruses. The protective effect was mediated by suppressing the production of major pro-inflammatory mediators. To further explore the anti-inflammatory properties of BPZE1, we investigated the effect of BPZE1 nasal pretreatment on two mouse models of allergic disease, allergic airway inflammation, and contact hypersensitivity (CHS). METHODS: Allergic reactions were induced in mice nasally pretreated with live attenuated BPZE1 bacteria using the ovalbumin (OVA)-induced allergic airway inflammation and dinitrochlorobenzene (DNCB)-induced CHS models. RESULTS: Prior BPZE1 nasal treatment suppressed OVA-induced lung inflammation and inflammatory cell recruitment and significantly reduced IgE levels and cytokine production. Similarly, BPZE1 nasal pretreatment markedly inhibited ear swelling, skin inflammation, and production of pro-inflammatory cytokines in the DNCB-induced CHS model. For both models, we showed that BPZE1 pretreatment does not affect the sensitization phase. Upon challenge, BPZE1 pretreatment selectively reduced the level of cytokines whose production is increased and did not affect the basal level of other cytokines. Together, our observations suggest that BPZE1 pretreatment specifically targets those cytokine-producing effector cells that are recruited and involved in the inflammatory reaction. CONCLUSION: Our study demonstrates the broad anti-inflammatory properties of the attenuated B. pertussis BPZE1 vaccine candidate and supports its development as a promising agent to prevent and/or treat allergic diseases.

Recombinant HBHA boosting effect on BCG-induced immunity against Mycobacterium tuberculosis infection.

Heterologous prime-boost regimens are effective strategies to promote long-term memory and strong cellular Th1 responses to Mycobacterium tuberculosis, when BCG is used in the priming step. Subcutaneous or intranasal boosting of BCG-vaccinated newborn mice with native heparin-binding haemagglutinin (nHBHA) significantly enhances protection against M. tuberculosis. However, nHBHA is characterized by a complex methylation pattern in its C-terminal domain, which is important for protective immunogenicity in primary vaccination. In this study we addressed the question whether boosting with recombinant, non-methylated HBHA (rHBHA) produced in Escherichia coli may enhance protection of BCG-primed newborn mice. We found that while subcutaneous rHBHA boosting enhanced protection of BCG-primed mice against intranasal M. tuberculosis infection both in spleen and lungs, enhanced protection against aerosol infection was only seen in the spleen (0.72 logs; P < 0.05) but not in the lungs. Thus, in BCG-primed mice the methylation of the C-terminal domain of HBHA is dispensable for the induction of enhanced protection in the lungs against intranasal but not aerosol infection, whereas it enhances protection in the spleen in both challenge models. This report thus provides evidence that rHBHA may be considered as a booster vaccine against disseminated tuberculosis.

Site-specific alterations in the B oligomer that affect receptor-binding activities and mitogenicity of pertussis toxin.

Pertussis toxin plays a major role in the pathogenesis of whooping cough and is considered an important constituent of vaccines against this disease. It is composed of five different subunits associated in a molar ratio 1S1:1S2:1S3:2S4:1S5. The S1 subunit is responsible for the ADP-ribosyltransferase activity of the toxin. The B moiety, composed of S2 through S5, recognizes and binds to the target cell receptors and has some ADP-ribosyltransferase-independent activities such as mitogenicity. Site-directed mutagenesis of subunits S2 and S3 allowed us to identify amino acid residues involved in receptor binding. Of all the modifications generated, the deletion of Asn 105 in S2 and of Lys 105 in S3 resulted in the more drastic reduction of binding to haptoglobin and CHO cells, respectively. A holotoxin carrying both deletions presented a mitogenicity reduced to an undetectable level. The combination of these B oligomer mutations with two substitutions in the S1 subunit led to the production of a toxin analog with reduced ADP-ribosyltransferase-dependent and -independent activities including mitogenicity. As shown by immunoprecipitation with various monoclonal antibodies, the mutant holotoxin was correctly assembled and antigenically similar to the native toxin. This toxin analog induced toxin-neutralizing antibodies at the same level as the holotoxin carrying only mutations in the S1 subunit, and may therefore be considered a useful candidate for the development of a new generation vaccine against whooping cough.

Identification of a heparin-binding hemagglutinin present in mycobacteria.

Adherence to mammalian host tissues is an important virulence trait in microbial pathogenesis, yet little is known about the adherence mechanisms of mycobacteria. Here, we show that binding of mycobacteria to epithelial cells but not to macrophages can be specifically inhibited by sulfated carbohydrates. Using heparin-Sepharose chromatography, a 28-kD heparin-binding protein was purified from culture supernatants and cell extracts of Mycobacterium bovis and Mycobacterium tuberculosis. This protein, designated heparin-binding hemagglutinin (HBHA), promotes the agglutination of rabbit erythrocytes, which is specifically inhibited by sulfated carbohydrates. HBHA also induce mycobacterial aggregation, suggesting that it can mediate bacteria-bacteria interactions as well. Hemagglutination, mycobacterial aggregation, as well as attachment to epithelial cells are specifically inhibited in the presence of anti-HBHA antibodies. Immunoelectron microscopy using anti-HBHA monoclonal antibodies revealed that the protein is surface exposed, consistent with a role in adherence. Immunoblot analyses using antigen-specific antibodies indicated that HBHA is different from the fibronectin-binding proteins of the antigen 85 complex and p55, and comparison of the NH2-terminal amino acid sequence of purified HBHA with the protein sequence data bases did not reveal any significant similarity with other known proteins. Sera from tuberculosis patients but not from healthy individuals were found to recognize HBHA, indicating its immunogenicity in humans during mycobacterial infections. Identification of putative mycobacterial adhesins, such as the one described in this report, may provide the basis for the development of new therapeutic and prophylactic strategies against mycobacterial diseases.

Role of DegP for two-partner secretion in Bordetella.

Sorting of proteins destined to the surface or the extracellular milieu is mediated by specific machineries, which guide the protein substrates towards the proper route of secretion and determine the compartment in which folding occurs. In gram-negative bacteria, the two-partner secretion (TPS) pathway is dedicated to the secretion of large proteins rich in beta-helical structure. The secretion of the filamentous haemagglutinin (FHA), a 230 kDa adhesin of Bordetella pertussis, represents a model TPS system. FHA is exported by the Sec machinery and transits through the periplasm in an extended conformation. From there it is translocated across the outer membrane by its dedicated transporter FhaC to finally fold into a long beta-helix at the cell surface in a progressive manner. In this work, we show that B. pertussis lacking the periplasmic chaperone/protease DegP has a strong growth defect at 37 degrees C, and the integrity of its outer membrane is compromised. While both phenotypes are significantly aggravated by the presence of FHA, the chaperone activity of DegP markedly alleviates the periplasmic stress. In vitro, DegP binds to non-native FHA with high affinity. We propose that DegP chaperones the extended FHA polypeptide in the periplasm and is thus involved in the TPS pathway.

Attenuated Bordetella pertussis vaccine strain BPZE1 modulates allergen-induced immunity and prevents allergic pulmonary pathology in a murine model.

BACKGROUND: Virulent Bordetella pertussis, the causative agent of whooping cough, exacerbates allergic airway inflammation in a murine model of ovalbumin (OVA) sensitization. A live genetically attenuated B. pertussis mucosal vaccine, BPZE1, has been developed that evokes full protection against virulent challenge in mice but the effect of this attenuated strain on the development of allergic responses is unknown. OBJECTIVE: To assess the influence of attenuated B. pertussis BPZE1 on OVA priming in a murine model of allergic airway inflammation. METHODS: Mice were challenged with virulent or attenuated strains of B. pertussis, and sensitized to allergen (OVA) at the peak of bacterial carriage. Subsequently, airway pathology, local inflammation and OVA-specific immunity were examined. RESULTS: In contrast to virulent B. pertussis, live BPZE1 did not exacerbate but reduced the airway pathology associated with allergen sensitization. BPZE1 immunization before allergen sensitization did not have an adjuvant effect on allergen specific IgE but resulted in a statistically significant decrease in airway inflammation in tissue and bronchoalveolar lavage fluid. BPZE1 significantly reduced the levels of OVA-driven IL-4, IL-5 and IL-13 but induced a significant increase in IFN-gamma in response to OVA re-stimulation. CONCLUSIONS: These data demonstrate that, unlike virulent strains, the candidate attenuated B. pertussis vaccine BPZE1 does not exacerbate allergen-driven airway pathology. BPZE1 may represent an attractive T-helper type 1 promoting vaccine candidate for eradication of whooping cough that is unlikely to promote atopic disease.

Pertussis toxin gene: nucleotide sequence and genetic organization.

The current pertussis vaccines, although efficacious, in some instances produce undesirable side effects. Molecular engineering of pertussis toxin, the major protective antigen, could provide a safer, new generation of vaccines against whooping cough. As a first critical step in the development of such a vaccine, the complete nucleotide sequence of the pertussis toxin gene was determined and the amino acid sequences of the individual subunits were deduced. All five subunits are coded by closely linked cistrons. A promoter-like structure was found in the 5'-flanking region, suggesting that the toxin is expressed through a polycistronic messenger RNA. The order of the cistrons is S1, S2, S4, S5, and S3. All subunits contain signal peptides of variable length. The calculated molecular weights of the mature subunits are 26,024 for S1, 21,924 for S2, 21,873 for S3, 12,058 for S4, and 11,013 for S5. Subunits S2 and S3 share 70% amino acid homology and 75% nucleotide homology. Subunit S1 contains two regions of eight amino acids homologous to analogous regions in the A subunit of both cholera and Escherichia coli heat labile toxins.

The coiled-coil N-terminal domain of the heparin-binding haemagglutinin is required for the humoral and cellular immune responses in mice.

Heparin-binding haemagglutinin (HBHA) is a 28-kDa mycobacterial adhesin, composed of three functional domains. Previous work has shown that the C-terminal methylated domain is important for adherence, and it is involved in protective T cell immunity in mouse models. However, the role of the coiled-coil N-terminal domain of HBHA in its overall immunogenic capacity remains elusive. Herein, a comparison of the antibody and cellular immune responses after subcutaneous and intranasal immunization of mice with HBHA (native and recombinant) revealed that the methylation pattern is important but not essential for this property. Subcutaneous immunization of mice with a truncated protein, rHBHADeltaC, which lacks the C-terminal methylated domain, was sufficient to trigger humoral and cellular immune responses to HBHA in mice. Altogether we provide evidence that the coiled-coil N-terminal domain is required for HBHA immunogenicity in vivo.

Structure-based mechanism of ligand binding for periplasmic solute-binding protein of the Bug family.

Bug proteins form a large family of periplasmic solute-binding proteins well represented in beta-proteobacteria. They adopt a characteristic Venus flytrap fold with two globular domains bisected by a ligand-binding cleft. The structures of two liganded Bug proteins have revealed that the family is specific for carboxylated solutes, with a characteristic mode of binding involving two highly conserved beta strand-beta turn-alpha helix motifs originating from each domain. These two motifs form hydrogen bonds with a carboxylate group of the ligand, both directly and via conserved water molecules, and have thus been termed the carboxylate pincers. In both crystallized Bug proteins, the ligands were found enclosed between the two domains and inaccessible to solvent, suggesting an inter-domain hinge-bending motion upon ligand binding. We report here the first structures of an open, unliganded Bug protein and of the same protein with a citrate ion bound in the open cavity. One of the ligand carboxylate groups is bound to one half of the carboxylate pincers by the beta strand-beta turn-alpha helix motif from domain 1, and the citrate ion forms several additional interactions with domain 1. The ligand is accessible to solvent and has very few contacts with domain 2. In this open, liganded structure, the second part of the carboxylate pincers originating from domain 2 is not stabilized by ligand binding, and a loop replaces the beta turn. In the unliganded structure, both motifs of the carboxylate pincers are highly mobile, and neither of the two beta turns is formed. Thus, ligand recognition is performed by domain 1, with the carboxylate group serving as an initial anchoring point. Stabilization of the closed conformation requires proper interactions to be established with domain 2, and thus domain 2 discriminates between productively and non-productively bound ligands.

Homologous and heterologous protection after single intranasal administration of live attenuated recombinant Bordetella pertussis.

While single-dose mucosal immunization is best achieved by the use of attenuated live microorganisms, attenuation generally results in decreased immunogenicity. We attenuated Bordetella pertussis by the deletion of the pertussis toxin gene. A single intranasal administration of this strain protected against subsequent challenge as well as did the parent strain and better than immunization with commercial vaccine. Unexpectedly, this attenuation resulted in increased immunogenicity against the protective antigen filamentous hemagglutinin (FHA). In addition, immunogenicity was also enhanced against the Schistosoma mansoni Sm28GST genetically fused to FHA, resulting in protection against the parasite, as characterized by a reduction in worm burden and egg charge, after a single intranasal administration. Thus, attenuated recombinant B. pertussis strains are promising vectors for the simultaneous protection against pertussis and heterologous diseases by a single intranasal administration.

Bordetella pertussis, molecular pathogenesis under multiple aspects.

Recent studies, including those based on genomics, have demonstrated that besides toxins and adhesins, Bordetella pertussis uses many additional virulence determinants. Most of them are part of the BvgAS regulon, although some, in particular iron-uptake systems, are independent of BvgAS. They are regulated by iron, although in one case, the production of a siderophore receptor could be linked to the BvgAS regulon.

Live pertussis vaccines: will they protect against carriage and spread of pertussis?

Pertussis is a severe respiratory disease that can be fatal in young infants. Its main aetiological agent is the Gram-negative micro-organism Bordetella pertussis. Vaccines against the disease have been in use since the 1950s, and global vaccination coverage has now reached more than 85%. Nevertheless, the disease has not been controlled in any country, and has even made a spectacular come-back in the industrialized world, where the first-generation whole-cell vaccines have been replaced by the more recent, less reactogenic, acellular vaccines. Several hypotheses have been proposed to explain these observations, including the fast waning of acellular vaccine-induced protection. However, recent mathematical modelling studies have indicated that asymptomatic transmission of B. pertussis may be the main reason for the current resurgence of pertussis. Recent studies in non-human primates have shown that neither whole-cell, nor acellular vaccines prevent infection and transmission of B. pertussis, in contrast to prior exposure. New vaccines that can be applied nasally to mimic natural infection without causing disease may therefore be useful for long-term control of pertussis. Several vaccine candidates have been proposed, the most advanced of which is the genetically attenuated B. pertussis strain BPZE1. This vaccine candidate has successfully completed a first-in-man phase I trial and was shown to be safe in young male volunteers, able to transiently colonize the nasopharynx and to induce antibody responses to B. pertussis antigens in all colonized individuals. Whether BPZE1 will indeed be useful to ultimately control pertussis obviously needs to be assessed by carefully conducted human efficacy trials.

Balance between Coiled-Coil Stability and Dynamics Regulates Activity of BvgS Sensor Kinase in Bordetella.

UNLABELLED: The two-component system BvgAS controls the expression of the virulence regulon of Bordetella pertussis. BvgS is a prototype of bacterial sensor kinases with extracytoplasmic Venus flytrap perception domains. Following its transmembrane segment, BvgS harbors a cytoplasmic Per-Arnt-Sim (PAS) domain and then a predicted 2-helix coiled coil that precede the dimerization-histidine-phosphotransfer domain of the kinase. BvgS homologs have a similar domain organization, or they harbor only a predicted coiled coil between the transmembrane and the dimerization-histidine-phosphotransfer domains. Here, we show that the 2-helix coiled coil of BvgS regulates the enzymatic activity in a mechanical manner. Its marginally stable hydrophobic interface enables a switch between a state of great rotational dynamics in the kinase mode and a more rigid conformation in the phosphatase mode in response to signal perception by the periplasmic domains. We further show that the activity of BvgS is controlled in the same manner if its PAS domain is replaced with the natural α-helical sequences of PAS-less homologs. Clamshell motions of the Venus flytrap domains trigger the shift of the coiled coil's dynamics. Thus, we have uncovered a general mechanism of regulation for the BvgS family of Venus flytrap-containing two-component sensor kinases. IMPORTANCE: The two-component system BvgAS of the whooping cough agent Bordetella pertussis regulates the virulence factors necessary for infection in a coordinated manner. BvgS is the prototype of a family of sensor kinase proteins found in major bacterial pathogens. When BvgS functions as a kinase, B. pertussis is virulent, and the bacterium shifts to an avirulent phase after BvgS senses chemicals that make it switch to phosphatase. Our goal is to decipher the signaling mechanisms of BvgS in order to understand virulence regulation in Bordetella, which may lead to new antimicrobial treatments targeting those two-component systems. We discovered that the activity of BvgS is regulated in a mechanical manner. A short region of the protein that precedes the enzymatic domain switches between two states in response to signal perception by other BvgS domains. This switch region is conserved among BvgS homologs, and thus, the regulation uncovered here will likely be relevant for the family.

In search of acceptable alternatives to the murine histamine sensitisation test (HIST): what is possible and practical?

The 'International Workshop on Alternatives to the Murine Histamine Sensitization Test for Acellular Pertussis Vaccines: In Search of Acceptable Alternatives to the Murine Histamine Sensitization Test (HIST): What is Possible and Practical?' was held on 4 and 5 March 2015 in London, United Kingdom. Participants discussed the results of the data generated from an international collaborative study (BSP114 Phase 2) sponsored by the European Directorate for the Quality of Medicines & Health Care (EDQM) to determine if a modified Chinese hamster ovary (CHO) cell-based clustering assay is a suitable alternative to replace HIST. Workshop participants agreed that protocol transferability demonstrated in the collaborative study indicates that a standardised CHO cell assay is adequate for measuring pure PTx in reference preparations. However, vaccine manufacturers would still need to demonstrate that the method is valid to detect or measure residual PTx in their specific adjuvanted products. The 2 modified CHO cell protocols included in the study (the Direct and the Indirect Methods) deserve further consideration as alternatives to HIST. Using the CHO cell assay, an in vitro alternative, for acellular pertussis (aP) vaccine batch release testing would reduce the number of animals used for aP vaccine safety testing. A strategic, stepwise adoption plan was proposed, in which the alternative test would be used for release purposes first, and then, once sufficient confidence in its suitable performance has been gained, its use would be extended to stability testing.

The modular architecture of bacterial response regulators. Insights into the activation mechanism of the BvgA transactivator of Bordetella pertussis.

Control of virulence factor expression in Bordetella pertussis is mediated by the products of the bvg operon. The BvgS membrane protein responds to certain environmental cues by activating the BvgA protein, which in turn modulates the expression of the target virulence factor genes. The BvgA and BvgS proteins are members of a large family of sensory transduction proteins called the two-component systems. We show that BvgA fusion proteins can activate transcription of a reporter gene containing the bvg promoter in Escherichia coli, and that this activity correlates with its ability to interact specifically with a recognition sequence in cognate promoters. Using homologies between BvgA and other bacterial response regulators as a guide, two BvgA truncation mutants were constructed and their transactivation and DNA-binding capacities were examined. We discovered that (1) DNA-binding activity is localized to the C-terminal half of BvgA, (2) sequence-specific DNA-binding is necessary, but not sufficient for transactivation, and (3) DNA-binding requires the last 20 amino acid residues at its carboxy terminus. A BvgA fusion protein lacking the receiver domain is inactive in transcriptional activation, but retains sequence-specific DNA-binding activity and forms multimeric complexes. We show that BvgA is able to utilize acetyl phosphate as a phosphoryl group donor and the instability of the covalent linkage at extremes of pH is consistent with an acyl phosphate group. Furthermore, the in vitro phosphorylated form of BvgA exhibits an enhanced capacity for binding DNA target sites, while a dephosphorylated form exhibits a limited capacity to bind these sites. We discuss the implications that these observations have on the mechanism by which BvgA is activated to a transcriptionally competent state.

Early cellular immune response to a new candidate mycobacterial vaccine antigen in childhood tuberculosis.

The search for novel vaccines against tuberculosis (TB) would benefit from in-depths knowledge of the human immune responses to Mycobacterium tuberculosis (Mtb) infection. Here, we characterised in a low TB incidence country, the immune responses to a new candidate vaccine antigen against TB, the heparin-binding haemagglutinin (HBHA), in young children in contact with an active TB case (aTB). Children with no history of BCG vaccination were compared to those vaccinated at birth to compare the initial immune responses to HBHA with secondary immune responses. Fifty-eight children with aTB and 76 with latent TB infection (LTBI) were included and they were compared to 90 non-infected children. Whereas Mtb-infected children globally secreted more interferon-gamma (IFN-γ) in response to HBHA compared to the non-infected children, these IFN-γ concentrations were higher in previously BCG-vaccinated compared to non-vaccinated children. The IFN-γ concentrations were similar in LTBI and aTB children, but appeared to differ qualitatively. Whereas the IFN-γ secretion induced by native methylated and recombinant non-methylated HBHA were well correlated for aTB, this was not the case for LTBI children. Thus, Mtb-infected young children develop IFN-γ responses to HBHA that are enhanced by prior BCG vaccination, indicating BCG-induced priming, thereby supporting a prime-boost strategy for HBHA-based vaccines. The qualitative differences between aTB and LTBI in their HBHA-induced IFN-γ responses may perhaps be exploited for diagnostic purposes.

Nasal vaccination using live bacterial vectors.

Live recombinant bacteria represent an attractive means to induce both mucosal and systemic immune responses against heterologous antigens. Several models have now been developed and shown to be highly efficient following intranasal immunization. In this review, we describe the two main classes of live recombinant bacteria: generally recognized as safe bacteria and attenuated strains derived from pathogenic bacteria. Among the latter, we have differentiated the bacteria, which do not usually colonize the respiratory tract from those that are especially adapted to respiratory tissues. The strategies of expression of the heterologous antigens, the invasiveness and the immunogenicity of the recombinant bacteria are discussed.

A new series of mycobacterial expression vectors for the development of live recombinant vaccines.

Recombinant BCG (bacillus Calmette-Guérin) is a promising candidate as a live vaccine delivery system. Thus far, however, only autoreplicative plasmids carrying the heterologous genes to be expressed in BCG, together with antibiotic-resistance genes, have been successfully used. This could potentially lead to the spreading of antibiotic resistance among other bacteria, and might therefore be unsafe for the environment. In this study, we present a series of three Escherichia coli-Mycobacteria shuttle vectors which enable expression and secretion of antigens without the use of antibiotic-resistance markers. All these plasmids confer mercury resistance to the host bacteria as the only selectable marker and contain a unique restriction site to allow for single-step in-frame cloning of open reading frames downstream from the Mycobacterium tuberculosis 85A antigen promoter and export signal. The system was used to express the free beta-subunit of human chorionic gonadotropin (hCG beta), a potential target of an immunotherapeutic vaccine.