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.     

A recombinant iron transport protein from Bordetella pertussis confers protection against Bordetella parapertussis

Whooping cough, which is caused by Bordetella pertussis and B. parapertussis, is a reemerging disease. New protective antigens are needed to improve the efficacy of current vaccines against both species. Using proteomic tools, it was here found that B. parapertussis expresses a homolog of AfuA, a previously reported new vaccine candidate against B. pertussis . It was found that this homolog, named AfuA_Bpp, is expressed during B. parapertussis infection, exposed on the surface of the bacteria and recognized by specific antibodies induced by the recombinant AfuA cloned from B. pertussis (rAfuA). Importantly, the presence of the O‐antigen, a molecule that has been found to shield surface antigens on B. parapertussis , showed no influence on antibody recognition of AfuA_Bpp on the bacterial surface. The present study further showed that antibodies induced by immunization with the recombinant protein were able to opsonize B. parapertussis and promote bacterial uptake by neutrophils. Finally, it was shown that this antigen confers protection against B. parapertussis infection in a mouse model. Altogether, these results indicate that AfuA is a good vaccine candidate for acellular vaccines protective against both causative agents of whooping cough.

     

The Bordetella type III secretion system: its application to vaccine development

B. pertussis is a causative agent of whooping cough (pertussis) in humans. Despite wide-scale vaccination in many countries, there is serious concern about pertussis as a re-emerging disease. Re-emergence of pertussis may be explained by several factors: the short duration of protection by the currently available acellular pertussis vaccine, an increase in asymptomatic adult carriers and expansion of strains with certain antigenic variations which are not covered by currently available vaccines. To develop safer and more efficacious vaccines which confer more prolonged protection, researchers are focusing on identification and characterization of new virulence factors. One candidate for protective antigens is the type III secretion system and its secreted proteins.     

Development of vaccines against pertussis caused by Bordetella holmesii using a mouse intranasal challenge model

Bordetella holmesii is recognized as the third causative agent of pertussis (whooping cough) in addition to Bordetella pertussis and Bordetella parapertussis. Pertussis caused by B. holmesii is not rare around the world. However, to date, there is no effective vaccine against B. holmesii. We examined the protective potency of pertussis vaccines available in Japan and vaccines prepared from B. holmesii. A murine model of respiratory infection was exploited to evaluate protective potency. No Japanese commercial pertussis vaccines were effective against B. holmesii. In contrast, a wBH vaccine and an aBH vaccine prepared from B. holmesii were both protective. Passive immunization with sera from mice immunized with aBH vaccine established protection against B. holmesii, indicating that B. holmesii‐specific serum antibodies might play an important role in protection. Immuno‐proteomic analysis with sera from mice immunized with aBH vaccine revealed that the sera recognized a BipA‐like protein of B. holmesii. An aBH vaccine prepared from a BipA‐like protein‐deficient mutant strain did not have a protective effect against B. holmesii. Taken together, our results suggest that the BipA‐like protein plays an important role in the protective efficacy of aBH vaccine.     

Acellular pertussis vaccination facilitates Bordetella parapertussis infection in a rodent model of bordetellosis

Despite over 50 years of population-wide vaccination, whooping cough incidence is on the rise. Although Bordetella pertussis is considered the main causative agent of whooping cough in humans, Bordetella parapertussis infections are not uncommon. The widely used acellular whooping cough vaccines (aP) are comprised solely of B. pertussis antigens that hold little or no efficacy against B. parapertussis. Here, we ask how aP vaccination affects competitive interactions between Bordetella species within co-infected rodent hosts and thus the aP-driven strength and direction of in-host selection. We show that aP vaccination helped clear B. pertussis but resulted in an approximately 40-fold increase in B. parapertussis lung colony-forming units (CFUs). Such vaccine-mediated facilitation of B. parapertussis did not arise as a result of competitive release; B. parapertussis CFUs were higher in aP-relative to sham-vaccinated hosts regardless of whether infections were single or mixed. Further, we show that aP vaccination impedes host immunity against B. parapertussis—measured as reduced lung inflammatory and neutrophil responses. Thus, we conclude that aP vaccination interferes with the optimal clearance of B. parapertussis and enhances the performance of this pathogen. Our data raise the possibility that widespread aP vaccination can create hosts more susceptible to B. parapertussis infection.     

Construction of Bordetella pertussis strains that overproduce genetically inactivated pertussis toxin.

Nontoxic analogs of pertussis toxin (PT), produced by in vitro mutagenesis of the tox operon, are immunogenic and protective against infection by Bordetella pertussis. The moderate levels of PT production by B. pertussis, however, make it the limiting antigen in the formulation of multicomponent, acellular, recombinant whooping cough vaccines. To increase production of the highly detoxified Lys9Gly129 PT analog by B. pertussis, additional copies of the mutated tox operon were integrated into the bacterial chromosome at the tox or fha locus by unmarked allelic exchange. Recombinant strains produced in this way secreted elevated levels of the PT analog proportional to gene dosage. The strains were stable during 10-liter fermentations, and yields of up to 80 mg of PT analog per liter were obtained under production-scale conditions. The nontoxic analog was purified and shown to be indistinguishable from material obtained from a B. pertussis strain that contained only a single copy of the toxLys9Gly129 operon. Such strains are therefore suitable for large-scale, industrial production of an acellular whooping cough vaccine containing a genetically detoxified PT analog.     

Construction of Bordetella pertussis strains that overproduce genetically inactivated pertussis toxin.

Nontoxic analogs of pertussis toxin (PT), produced by in vitro mutagenesis of the tox operon, are immunogenic and protective against infection by Bordetella pertussis. The moderate levels of PT production by B. pertussis, however, make it the limiting antigen in the formulation of multicomponent, acellular, recombinant whooping cough vaccines. To increase production of the highly detoxified Lys9Gly129 PT analog by B. pertussis, additional copies of the mutated tox operon were integrated into the bacterial chromosome at the tox or fha locus by unmarked allelic exchange. Recombinant strains produced in this way secreted elevated levels of the PT analog proportional to gene dosage. The strains were stable during 10-liter fermentations, and yields of up to 80 mg of PT analog per liter were obtained under production-scale conditions. The nontoxic analog was purified and shown to be indistinguishable from material obtained from a B. pertussis strain that contained only a single copy of the toxLys9Gly129 operon. Such strains are therefore suitable for large-scale, industrial production of an acellular whooping cough vaccine containing a genetically detoxified PT analog.     

The effect of pertussis whole cell and acellular vaccines on pulmonary immunology in an aerosol challenge model

Recent clinical trials have shown that the new generation of acellular pertussis vaccines (Pa) can confer protection against whooping cough with negligible adverse reactions. We have compared the effects of pertussis whole cell and acellular vaccines on pulmonary immune responses after aerosol challenge in a murine model of infection. Mice were vaccinated with PBS, Pw or Pa and challenged with Bordetella pertussis by the aerosol route. Cytokine gene expression was analysed from lung tissue and cells; lung lymphocytes were re-stimulated in vitro and cytokines produced measured. The results obtained are consistent with the proposal that a strong Th-1 response is associated with bacterial clearance in both the non-vaccinated and Pw vaccinated mice. The acellular vaccine treated mice cleared the bacterial challenge (with an intermediate efficacy) in the presence of low levels of any of the cytokines assessed. This suggests that Pa protects via a Th-2 independent mechanism.     

Immunological surveys of antibodies against B. pertussis and B. parapertussis in some African and Asian countries.

Immunological surveys in African and Asian countries showed a different degree of herd immunity in the respective countries and the circulation of various types of B. pertussis. Antibodies against B. parapertussis, the second aertiological agent of whooping cough, were found in all countries in all age groups. Before planning any vaccination program, attention should be paid to the vaccine concerning the content of all types of B. pertussis, as was shown in the results of the testing of different vaccines used in Mongolia and Algeria. It is also possible to estimate the age limit for vaccination and thus economic use of the vaccine. The contemporary state of reporting whooping cough cases is very unsatisfactory.     

Phosphorylation chemistry of the Bordetella PlrSR TCS and its contribution to bacterial persistence in the lower respiratory tract

Bordetella species cause lower respiratory tract infections in mammals. B. pertussis and B. bronchiseptica are the causative agents of whooping cough and kennel cough, respectively. The current acellular vaccine for B. pertussis protects against disease but does not prevent transmission or colonization. Cases of pertussis are on the rise even in areas of high vaccination. The PlrSR two-component system, is required for persistence in the mouse lung. A partial plrS deletion strain and a plrS H521Q strain cannot survive past 3 days in the lung, suggesting PlrSR works in a phosphorylation-dependent mechanism. We characterized the biochemistry of B. bronchiseptica PlrSR and found that both proteins function as a canonical two-component system. His521 was essential and Glu522 was critical for PlrS autophosphorylation. Asn525 was essential for phosphatase activity. The PAS domain was critical for both PlrS autophosphorylation and phosphatase activities. PlrS could both phosphotransfer to and exert phosphatase activity toward PlrR. Unexpectedly, PlrR formed a tetramer when unphosphorylated and a dimer upon phosphorylation. Finally, we demonstrated the importance of PlrS phosphatase activity for persistence within the murine lung. By characterizing PlrSR we hope to guide future in vivo investigation for development of new vaccines and therapeutics.     

Live attenuated B. pertussis as a single-dose nasal vaccine against whooping cough

Pertussis is still among the principal causes of death worldwide, and its incidence is increasing even in countries with high vaccine coverage. Although all age groups are susceptible, it is most severe in infants too young to be protected by currently available vaccines. To induce strong protective immunity in neonates, we have developed BPZE1, a live attenuated Bordetella pertussis strain to be given as a single-dose nasal vaccine in early life. BPZE1 was developed by the genetic inactivation or removal of three major toxins. In mice, BPZE1 was highly attenuated, yet able to colonize the respiratory tract and to induce strong protective immunity after a single nasal administration. Protection against B. pertussis was comparable to that induced by two injections of acellular vaccine (aPV) in adult mice, but was significantly better than two administrations of aPV in infant mice. Moreover, BPZE1 protected against Bordetella parapertussis infection, whereas aPV did not. BPZE1 is thus an attractive vaccine candidate to protect against whooping cough by nasal, needle-free administration early in life, possibly at birth.     

Etiology, clinical manifestations, and epidemiology in community-acquired respiratory infection in children in the Khabarovsk region

Clinico-epidemiological analysis and etiological verification of the outbreak of respiratory infection among school children in a rural district of the Khabarovsk territory, registered in spring 1997, were made. According to clinical signs, one-third of the patients had whooping cough, while the rest of the children exhibited the signs of respiratory infection with the symptoms of longering bronchitis. A half of the children had not been vaccinated against whooping cough, as they had been given injections of adsorbed DT vaccine with reduced antigen content. Etiologically, the diagnosis of whooping cough was confirmed in 57% of the patients with 47.4% of them having Bordetella pertussis monoinfection and 52.6% having mixed infection, mainly in combination with chlamydiosis. Whooping cough took an abnormal course under these circumstances. Treatment with erythromycin produced a good effect.     

Identification of immunodominant epitopes in the filamentous hemagglutinin of Bordetella pertussis

The filamentous hemagglutinin (FHA) of Bordetella pertussis is a principal adhesin, which plays a key role in the colonization of the upper respiratory tract. FHA is also a protective antigen, which has been incorporated in the new generation of acellular vaccines against whooping cough. The protein is synthesized as a large 367-kDa precursor, which is then processed into a 220-kDa secreted polypeptide. To optimize the use of this protein for vaccine purposes it would be helpful to define the regions encompassing immunodominant epitopes. Twelve recombinant plasmids have been generated encoding fusion proteins between fragments of the matured-secreted 220-kDa form of FHA and the vector-encoded phage MS2 polymerase. Protein extracts of the resulting recombinant clones have been tested for reactivity with sera from 20 patients convalescent from whooping cough, and two human standard sera. The results indicate the presence of an immunodominant B cell epitope in the polypeptide coded by a 1-kb DNA fragment encompassing positions 5781-6800 of the published sequence. These results suggest that the identified fragment should be conserved in the formulation of vaccines against pertussis.     

Acellular pertussis booster in adolescents induces Th1 and memory CD8+ T cell immune response

In a number of countries, whole cell pertussis vaccines (wcP) were replaced by acellular vaccines (aP) due to an improved reactogenicity profile. Pertussis immunization leads to specific antibody production with the help of CD4(+) T cells. In earlier studies in infants and young children, wcP vaccines selectively induced a Th1 dominated immune response, whereas aP vaccines led to a Th2 biased response. To obtain data on Th1 or Th2 dominance of the immune response in adolescents receiving an aP booster immunization after a wcP or aP primary immunization, we analyzed the concentration of Th1 (IL-2, TNF-α, INF-γ) and Th2 (IL-4, IL-5, IL-10) cytokines in supernatants of lymphocyte cultures specifically stimulated with pertussis antigens. We also investigated the presence of cytotoxic T cell responses against the facultative intracellular bacterium Bordetella pertussis by quantifying pertussis-specific CD8(+) T cell activation following the aP booster immunization. Here we show that the adolescent aP booster vaccination predominantly leads to a Th1 immune response based on IFNgamma secretion upon stimulation with pertussis antigen, irrespective of a prior whole cell or acellular primary vaccination. The vaccination also induces an increase in peripheral CD8(+)CD69(+) activated pertussis-specific memory T cells four weeks after vaccination. The Th1 bias of this immune response could play a role for the decreased local reactogenicity of this adolescent aP booster immunization when compared to the preceding childhood acellular pertussis booster. Pertussis-specific CD8(+) memory T cells may contribute to protection against clinical pertussis.     

Restriction analysis of Bordetella pertussis DNA isolated from patients with whooping cough in 1968 and 1995-98 and B. pertussis used for production of national vaccine strains

The genotypic and serotypic analysis of B. pertussis strains isolated from the nasopharynx of children with whooping cough in the years 1968 and 1995-98 and B. pertussis vaccine strains was the aim of this study. The genotyping of the examined strains was done by electrophoretic division of DNA in pulsed field. The 3 types (A, B, C) and 2 subtypes (A1 and A2) of DNA restriction patterns were determined for the B. pertussis strains isolated in 1968. The 2 types (D and E) and 10 subtypes (D1-D10) of DNA restriction patterns were identified for B. pertussis strains from the years 1995-98. The DNA restriction patterns of B. pertussis strains isolated in the years 1968 and 1995-98 were not identical what was the evidence of the fact that in the sixties and nineties whooping cough was caused by different B. pertussis clones. The different DNA profiles were also observed for vaccine strains as well as for vaccine strains and current isolates. Differences in DNA patterns of vaccine strains and B. pertussis strains isolated in the years 1995-98 indicated a relationship possibility in some cases while lack of relationship between these strains in other cases. Serotyping of the examined B. pertussis strains was performed by the agglutination method with the sera against B. pertussis agglutinogens 1, 2 and 3. Most strains--15 (75%) isolated in 1968 possessed only agglutinogens 1 and 3. Serotype 1, 2, 3 was most frequently observed among isolates from the years 1995-98. This study indicates the expediency of periodical change of B. pertussis vaccine strains in the aspect of whooping cough resurgence in the years 1994-95 and 1997-98.     

A Novel IgM‐capture enzyme‐linked immunosorbent assay using recombinant Vag8 fusion protein for the accurate and early diagnosis of Bordetella pertussis infection

An ELISA that measures anti‐PT IgG antibody has been used widely for the serodiagnosis of pertussis; however, the IgG‐based ELISA is inadequate for patients during the acute phase of the disease because of the slow response of anti‐PT IgG antibodies. To solve this problem, we developed a novel IgM‐capture ELISA that measures serum anti‐Bordetella pertussis Vag8 IgM levels for the accurate and early diagnosis of pertussis. First, we confirmed that Vag8 was highly expressed in all B. pertussis isolates tested (n = 30), but little or none in other Bordetella species, and that DTaP vaccines did not induce anti‐Vag8 IgG antibodies in mice (i.e. the antibody level could be unaffected by the vaccination). To determine the immune response to Vag8 in B. pertussis infection, anti‐Vag8 IgM levels were compared between 38 patients (acute phase of pertussis) and 29 healthy individuals using the anti‐Vag8 IgM‐capture ELISA. The results revealed that the anti‐Vag8 IgM levels were significantly higher in the patients compared with the healthy individuals (P < 0.001). ROC analysis also showed that the anti‐Vag8 IgM‐capture ELISA has higher diagnostic accuracy (AUC, 0.92) than a commercial anti‐PT IgG ELISA kit. Moreover, it was shown that anti‐Vag8 IgM antibodies were induced earlier than anti‐PT IgG antibodies on sequential patients’ sera. These data indicate that our novel anti‐Vag8 IgM‐capture ELISA is a potentially useful tool for making the accurate and early diagnosis of B. pertussis infection.     

Production and characterization of recombinant pertactin, fimbriae 2 and fimbriae 3 from Bordetella pertussis

Background  

Bordetella pertussis is a causative agent of pertussis or whooping cough in humans. Pertactin (Prn), fimbriae 2 (Fim2) and fimbriae 3 (Fim3) of B. pertussis are important virulence factors and immunogens which have been included in some acellular pertussis vaccines. In this present study, we cloned, expressed and purified Prn, Fim2 and Fim3, respectively. The immunogeniCity and protective efficacy of the three recombinant proteins (rPrn, rFim2 and rFim3) were investigated in mouse model.     

An international collaborative study of the effect of active pertussis toxin on the modified Kendrick test for acellular pertussis vaccines

Speculation that the Japanese modified intra-cerebral challenge assay, which is used in several countries for control of acellular pertussis vaccines, depends on the presence of small amounts of active pertussis toxin led to an assumption that it may not be appropriate for highly toxoided or genetically detoxified vaccines. Consequently, at the recommendation of a World Health Organisation AD Hoc Working Group on mouse protection models for testing and control of acellular pertussis vaccine, the effect of pertussis toxin on the modified intra-cerebral challenge assay (modified Kendrick, MICA) was evaluated in an international collaborative study. Results of this study showed that for genetically detoxified vaccines both with and without active pertussis toxin the MICA clearly distinguished mice vaccinated with acellular vaccines from unvaccinated mice and gave a significant dose–response relationship. However, vaccine samples containing active pertussis toxin (5 or 50 ng/single human dose) appeared to be more potent than the equivalent sample without active pertussis toxin. Similar results were also given by two respiratory infection models (intranasal and aerosol) included in the study. The results also indicated that the effect of pertussis toxin may vary depending on mouse strain.     

O Antigen Allows B. parapertussis to Evade B. pertussis Vaccine–Induced Immunity by Blocking Binding and Functions of Cross-Reactive Antibodies

Although the prevalence of Bordetella parapertussis varies dramatically among studies in different populations with different vaccination regimens, there is broad agreement that whooping cough vaccines, composed only of B. pertussis antigens, provide little if any protection against B. parapertussis. In C57BL/6 mice, a B. pertussis whole-cell vaccine (wP) provided modest protection against B. parapertussis, which was dependent on IFN-γ. The wP was much more protective against an isogenic B. parapertussis strain lacking O-antigen than its wild-type counterpart. O-antigen inhibited binding of wP–induced antibodies to B. parapertussis, as well as antibody-mediated opsonophagocytosis in vitro and clearance in vivo. aP–induced antibodies also bound better in vitro to the O-antigen mutant than to wild-type B. parapertussis, but aP failed to confer protection against wild-type or O antigen–deficient B. parapertussis in mice. Interestingly, B. parapertussis–specific antibodies provided in addition to either wP or aP were sufficient to very rapidly reduce B. parapertussis numbers in mouse lungs. This study identifies a mechanism by which one pathogen escapes immunity induced by vaccination against a closely related pathogen and may explain why B. parapertussis prevalence varies substantially between populations with different vaccination strategies.     

The Bordetella pertussis Bps polysaccharide enhances lung colonization by conferring protection from complement‐mediated killing

Bordetella pertussis is a human‐restricted Gram‐negative bacterial pathogen that causes whooping cough or pertussis. Pertussis is the leading vaccine preventable disease that is resurging in the USA and other parts of the developed world. There is an incomplete understanding of the mechanisms by which B. pertussis evades killing and clearance by the complement system, a first line of host innate immune defence. The present study examined the role of the Bps polysaccharide to resist complement activity in vitro and in the mouse respiratory tract. The isogenic bps mutant strain containing a large non‐polar in‐frame deletion of the bpsA–D locus was more sensitive to serum and complement mediated killing than the WT strain. As determined by Western blotting, flow cytometry and electron microscopic studies, the heightened sensitivity of the mutant strain was due to enhanced deposition of complement proteins and the formation of membrane attack complex, the end‐product of complement activation. Bps was sufficient to confer complement resistance as evidenced by a Bps‐expressing Escherichia coli being protected by serum killing. Additionally, Western blotting and flow cytometry assays revealed that Bps inhibited the deposition of complement proteins independent of other B. pertussis factors. The bps mutant strain colonized the lungs of complement‐deficient mice at higher levels than that observed in C57Bl/6 mice. These results reveal a previously unknown interaction between Bps and the complement system in controlling B. pertussis colonization of the respiratory tract. These findings also make Bps a potential target for the prevention and therapy of whooping cough.