Effectiveness of the whole-cell pertussis vaccine produced in Poland against different Bordetella parapertussis isolates in the mouse intranasal challenge model

The aim of the study was to evaluate the effectiveness of the whole-cell pertussis vaccine produced locally and routinely used in Poland in the elimination of Bordetella parapertussis strains from the lungs and trachea of a mouse model. We found that the average protective effect against B. parapertussis in the lungs of mice immunized with the whole-cell pertussis vaccine (DTwP) was significantly higher than in animals immunized with the acellular pertussis vaccine (DTaP). The effectiveness of B. parapertussis elimination rates from the lungs of DTwP-immunized mice, depending on the strain used as a challenge, was found to be 1.2-3.0 times or 3.1-7.0 times lower than against Bordetella. pertussis Tohama I or vaccine B. pertussis 606/67 isolates, respectively. Our results show that the locally produced DTwP vaccine is able to protect against B. parapertussis isolates; however, the level of protection and course of B. parapertussis infection in the lungs and trachea seems to be strain specific.     

The importance of pertussis in older adults: A growing case for reviewing vaccination strategy in the elderly

Pertussis or whooping cough is increasingly being shown to be a respiratory infection affecting the elderly and a significant percentage of older people infected with Bordetella pertussis experience considerable morbidity and even mortality. However, current knowledge of burden of disease is limited largely to passive surveillance data with little well-designed active surveillance to better ascertain the true burden of pertussis in the elderly, to inform vaccination strategies. The current review aims to identify gaps in knowledge to inform policy considerations relating to pertussis vaccination among the elderly.     

Identification of a new protective antigen of Bordetella pertussis

Antigenic proteins whose expression is induced under iron starvation, an environmental condition that bacterial pathogens have to face during colonization, might be potential candidates for improved vaccine. By mean of immune proteomics we identified novel antigens of Bordetella pertussis maximally expressed under iron limitation. Among them, Bp1152 (named as IRP1-3) showed a particularly strong reaction with human IgG purified from pooled sera of pertussis-infected individuals. Computer analysis showed IRP1-3 as a dimeric membrane protein potentially involved in iron uptake. Experimental data revealed the surface-exposure of this protein and showed its increase under iron starvation to be independent of bacterial virulence phase. Immunization of mice with the recombinant IRP1-3 resulted in a strong antibody response. These antibodies not only recognized the native protein on bacterial surface but also promote effective bacterial phagocytosis by human PMN, a key protecting activity against this pathogen. Accordingly, IRP1-3 proved protective against B. pertussis infection in mouse model. Expression of IRP1-3 was found conserved among clinical isolates of B. pertussis and positively regulated by iron starvation in these strains. Taken together these results suggest that this protein might be an interesting novel vaccine candidate.     

Dual mechanism of protection by live attenuated Bordetella pertussis BPZE1 against Bordetella bronchiseptica in mice

Bordetella bronchiseptica, a gram-negative bacterium, causes chronic respiratory tract infections in a wide variety of mammalian hosts, including man, and no human vaccine is currently available. Acellular pertussis vaccines protect poorly against B. bronchiseptica, although they contain cross-reactive antigens. We have recently developed Bordetella pertussis BPZE1, a novel, live attenuated pertussis vaccine, currently completing phase I clinical trials in humans, and found that it protects against both B. pertussis and Bordetella parapertussis in mice. Here, we show that a single nasal administration of BPZE1 protects mice against lethal infection with B. bronchiseptica. After challenge, the vaccinated animals displayed markedly reduced lung inflammation and tissue damage, decreased neutrophil infiltration and increased levels of CD4(+)CD25(+)FoxP3(+) regulatory T cells in the lungs compared to non-immunized mice. Depletion of these cells abolished BPZE1-induced protection, indicating that BPZE1 protects against lethal inflammation through the recruitment of regulatory T cells. In addition, the B. bronchiseptica load was significantly decreased in the vaccinated animals. Using passive transfer experiments, protection was found to be essentially cell mediated, and BPZE1-induced Th1 and Th17 T cells recognize whole B. bronchiseptica extracts, although the participation of antibodies in protection cannot be discounted. Thus, a single administration of BPZE1 can confer protection against B. bronchiseptica in mice by a dual mechanism.     

Bordetella pertussis vaccine strains and circulating isolates in Serbia

In Serbia, whole cell pertussis vaccine was introduced in 1957. Current composition of the vaccine has been used since 1985 and contains four autochthonous strains of Bordetella pertussis isolated from 1957 to 1984. To monitor changes in bacterial population, 70 isolates collected from 1953 to 2000 were studied together with the vaccine strains. The methods included serotyping of fimbriae (Fim), genotyping of pertactin (prn) and pertussis toxin S1 subunit (ptxA), and pulsed-field gel electrophoresis analysis. Shift from ptxA2 to ptxA1 has been observed in isolates since the late of 1960s. All isolates from 1980 to 1984 harbored ptxA1. Re-appearance of the ptxA2 allele followed an addition of the two strains harboring ptxA1 in the vaccine in 1985. The allele prn1 was predominant among the Serbian isolates, though prn3 and prn11 have been detected since 1981 and 1984. The allele prn2 was found only in two strains isolated in 2000. Serotype Fim2.3 disappeared before 1980 and serotype Fim2 became predominant since then. The Serbian vaccine strains showed differences in ptxA and prn. The results of this present study indicate that the B. pertussis population in Serbia is different from other vaccinated populations and that this difference may be related to the vaccine used.     

Pertussis before and after the introduction of acellular pertussis vaccines in Finland

In Finland, the whole-cell pertussis vaccine was replaced with acellular pertussis vaccine in the national immunisation schedule in 2005. Adolescent booster vaccinations were also included in the programme. The aim of this study was to evaluate the effects of these changes on the epidemiology and strain characteristics of Bordetella pertussis. From the national register, we first analysed all the laboratory diagnosed cases during the study years in 1999–2006. The major pool of the 6876 cases was among adolescents and adults. After the change of the programme and the introduction of the adolescent boosters, a general reduction of the incidence was noticed but this might be related to the natural epidemic cycles of pertussis. Secondly, a questionnaire was sent to the families of the 517 young children (<2 years of age) with registered, laboratory confirmed pertussis diagnosed during the study years. Of these, 319 (62%) participated the study. Forty-five percents of the cases in this cohort were younger than 3 months, the age of the first pertussis immunisation in schedule. Only 4% of the children in vaccination age were totally unimmunised. Thirdly, isolates of B. pertussis were analysed and found to differ from the used whole-cell pertussis vaccine strains by their prn, ptxA and PFGE profiles. However, no significant differences were found between the strains from patients with different immunisation status or age. Despite marked changes in the virulence genes and the genomes of the circulating B. pertussis strains have occurred, the epidemiological data from the national reporting system indicates that the whole-cell and acellular vaccines still protect against pertussis, but the results stress the importance of early primary immunisations and the need for booster immunisations.     

Acellular pertussis vaccine based on outer membrane vesicles capable of conferring both long-lasting immunity and protection against different strain genotypes

Despite high vaccination coverage rates, pertussis continues to be a global concern, with increased incidence widely noted. The current pertussis epidemiologic situation has been mainly attributed to waning immunity and pathogen adaptation. To improve the disease control, a new generation of vaccines capable to overcome those weaknesses associated to the current vaccines need to be developed. Previously we have demonstrated that the outer membrane vesicles obtained from the recombinant Bordetella pertussis strain expressing PagL enzyme (OMVs_BpPagL) are good vaccine candidates to protect against pertussis. In this work the OMVs_BpPagL formulated with diphtheria and tetanus toxoids (Tdap_OMVsBpPagL) was used to evaluate its capacity to offer protection against Argentinean clinical isolates and to induce long-term immunity. To these aims BALB/c mice were immunized with Tdap_OMVsBpPagL and challenged with sublethal doses of the clinical isolate Bp106 selected as a representative circulating isolate. Comparisons with a current commercial Tdap vaccine used at a dose in which pertussis toxin level was equivalent to that of Tdap_OMVsBpPagL were performed. With the normalized doses of both vaccines we observed that Tdap_OMVsBpPagL protected against the clinical isolate infection, whereas current commercial Tdap vaccine showed little protection against such pathogen. Regarding long-term immunity we observed that the Tdap_OMVsBpPagL protective capacity against the recommended WHO reference strain persisted at least 9 months. In agreement with these results Tdap_OMVsBpPagL induced Th1 and Th2 immune response. In contrast, commercial Tdap induced Th2 but weak Th1 responses. All results presented here showed that Tdap_OMVsBpPagL is an interesting formulation to be considered for the development of novel acellular multi-antigen vaccine.     

First report and detailed characterization of B. pertussis isolates not expressing pertussis toxin or pertactin

Bordetella pertussis isolates not expressing Pertussis Toxin (PT) or Pertactin (PRN) have been collected, for the first time in 2007, in France, a highly vaccinated country with acellular vaccines. Non-expression was due to deletion of the entire ptx locus, to IS481 insertion in the prn gene or deletion of a part of this gene. Genome sequencing does not indicate any regions of differences when compared to other circulating isolates. It nevertheless shows some sequence differences and an increased number of repeated sequences. The infant infected by the isolate not expressing pertussis toxin, did not present hyperlymphocytosis. All isolates were found less pathogen in animal or cellular models; their circulation raises the problem of clinical and biological diagnoses.     

Control of pertussis—Lessons learnt from a 10-year surveillance programme in Sweden

Sweden was the only country in the world without any general pertussis vaccination when acellular pertussis (aP) vaccines were introduced. Since 1996 aP vaccines are given at the ages of 3, 5 and 12 months, with a 99% coverage, and until 2007 without a later booster. The long-term effects of aP vaccines, monitored within an enhanced surveillance project, were discussed at an international workshop in Stockholm in November 2008. The unique Swedish experience demonstrates that aP vaccines are capable of achieving the primary goal of a national vaccination programme, i.e., to significantly reduce the burden of pertussis in pre-school children. Throughout the 10-year surveillance period the highest age-specific incidence was reported in unvaccinated infants or those who had received only one dose, with most hospitalisations in this age group and eight deaths among unvaccinated infants. Complementary strategies are needed to achieve further reduction in morbidity from circulation of Bordetella pertussis.     

Antibody levels against B. pertussis in neonates measured in dried blood spots

Aim

We designed this study to investigate if immunoglobuline G-Pertussis toxin (IgG-PT) against Bordetella pertussis in umbilical cord blood can reliably be determined in dried blood spots on filter paper (Guthrie) cards.

Patients and methods

We prospectively included 129 mothers and their newborns born in a general hospital in the Netherlands. The relation between IgG-PT against B. pertussis from the umbilical cord measured in dried blood spots (Guthrie card) and in serum samples was studied by means of a Bland–Altman graph, using regression analysis to evaluate the level of agreement of both measurement methods.

Results

IgG-PT in Guthrie cards show a high coefficient of correlation with IgG-PT in serum samples from the umbilical cord when calibrated against blood spot calibrators (p < 0.05).

Conclusion

Maternal IgG-PT against B. pertussis measured in cord blood applied to Guthrie cards and calibrated against blood spot calibrators show good agreement with measurement of IgG-PT in cord serum. This offers new perspectives for future studies concerning B. pertussis antibodies.     

Development of live attenuated Bordetella pertussis strains expressing the universal influenza vaccine candidate M2e

The attenuated Bordetella pertussis BPZE1 vaccine strain represents an attractive platform for the delivery of heterologous vaccine candidates via the nasal route. The filamentous hemagglutinin (FHA) has been used to secrete or expose the foreign antigens at the bacterial surface. In this study, one, two and three copies of the Cys-containing ectodomain of matrix protein 2 (M2e) from influenza A virus were genetically fused to full length FHA and expressed in BPZE1. The secretion efficacy of the FHA-(M2e)_1,2,3 chimera in the extracellular milieu and the ability of the recombinant bacteria to colonize the mouse lungs inversely correlated with the number of M2e copies fused to FHA. Nevertheless FHA-(M2e)₃-producing bacteria (BPLR3) triggered the highest systemic anti-M2e antibody response upon nasal administration to BALB/c mice. Nasal immunization with BPLR3 bacteria resulted in a significant reduction in the viral loads upon challenge with H1N1/PR8 influenza A virus, but did not improve the survival rate compared to BPZE1-immunized mice. Furthermore, since previous work reported that disulfide bond formation in Cys-containing passenger antigens affects the secretion efficacy of the FHA chimera, the dsbA gene encoding a periplasmic disulfide isomerase was deleted in the FHA-(M2e)₃-producing strain. Despite improving significantly the secretion efficacy of the FHA-(M2e)₃ chimera, the dsbA deletion did not result in higher anti-M2e antibody titers in mice, due to impaired bacterial fitness and colonization ability.     

Pertactin deficient Bordetella pertussis present a better fitness in mice immunized with an acellular pertussis vaccine

Bordetella pertussis is the etiologic agent of whooping cough and has been the target of vaccination for over fifty years. The latest strategies include the use of acellular pertussis vaccines that induce specific immunity against few virulence factors amongst which pertactin is included in three and five component acellular pertussis vaccines. Recently, it has been reported that B. pertussis clinical isolates loose the production of this adhesin in regions reaching high vaccine coverage with vaccines targeting this virulence factor. We here demonstrate that isolates not producing pertactin are capable of sustaining longer infection as compared to pertactin producing isolates in an in vivo model of acellular pertussis immunization. Loosing pertactin production might thus provide a selective advantage to these isolates in this background, which could account for the upraise in prevalence of these pertactin deficient isolates in the population.     

Outer membrane vesicles obtained from Bordetella pertussis Tohama expressing the lipid A deacylase PagL as a novel acellular vaccine candidate

In an effort to devise a safer and effective pertussis acelullar vaccine, outer membrane vesicles (OMVs) were engineered to decrease their endotoxicity. The pagL gene from Bordetella bronchiseptica, which encodes a lipid A 3-deacylase, was expressed in Bordetella pertussis strain Tohama I. The resulting OMVs, designated OMVs_BpPagL, contain tetra- instead of penta-acylated LOS, in addition to pertussis surface immunogens such as pertactin and pertussis toxin, as the wild type OMVs. The characterized pertussis OMVs_BpPagL were used in murine B. pertussis intranasal (i.n.) challenge model to examine their protective capacity when delivered by i.n. routes. Immunized BALB/c mice were challenged with sublethal doses of B. pertussis. Significant differences between immunized animals and the PBS treated group were observed (p < 0.001). Adequate elimination rates (p < 0.005) were observed in mice immunized either with OMVs_BpPagL and wild type OMVs. All OMV preparations tested were non toxic according to WHO criteria; however, OMVs_BpPagL displayed almost no weight loss at 3 days post administration, indicating less toxicity when compared with wild type OMVs. Induction of IL6- and IL1-expression in lung after i.n. delivery as well as neutrophil recruitment to airways showed coincident results, with a lower induction of the proinflammatory cytokines and lower recruitment in the case of OMVs_BpPagL compared to wild type OMVs.Given their lower endotoxic activity and retained protective capacity in the mouse model, OMVs_BpPagL obtained from B. pertussis seem as interesting candidates to be considered for the development of novel multi-antigen vaccine.     

An investigation into the cause of the 1983 whooping cough epidemic in the Netherlands

Despite more than 50 years of vaccination, whooping cough is still an endemic disease in the Netherlands with regular epidemic outbreaks. In the last 20 years, two periods of increased notifications were observed. The causes of the increased notifications in the first period, from 1983 to 1987, are contentious. At the time it was suggested to be a surveillance artifact, caused by changes in diagnostic procedures and increased awareness. An alternative explanation, a reduction in the vaccine dose, was downplayed at the time. The aim of this study was to reinvestigate the causes of the increased notifications by identifying changes in the Bordetella pertussis population. B. pertussis strains, isolated from 1965 to 1992, were characterized by means of fimbrial serotyping, multiple-locus sequence typing of virulence genes (MLST) and multiple-locus variable-number tandem repeat analysis (MLVA). Shifts in fimbrial serotypes and MLVA types were associated with changes in vaccine dose and increased number of notifications. One to three years after lowering of the vaccine dose, the predominant fimbrial serotype changed from Fim3 to Fim2, and the reverse trend was observed when the vaccine dose was increased. Significantly, changes in fimbrial serotypes were evident at least seven years before the increase in notifications. Our results provide evidence that the change in vaccine dose affected host immunity and, consequently, contributed to an increase in pertussis morbidity. Further, we show that MLVA and fimbrial serotyping of strains can be used as early warning for pertussis epidemics.     

Bordetella pertussis Strain Lacking Pertactin and Pertussis Toxin

A Bordetella pertussis strain lacking 2 acellular vaccine immunogens, pertussis toxin and pertactin, was isolated from an unvaccinated infant in New York State in 2013. Comparison with a French strain that was pertussis toxin–deficient, pertactin wild-type showed that the strains carry the same 28-kb deletion in similar genomes.     

Comparison of recombinant and native pertactin of Bordetella pertussis

Bordetella pertussis pertactin (Prn) is an important attachment factor and protective immunogen, which serves as a component in most acellular pertussis vaccines (APVs). Here, we over-expressed recombinant Prn (r-Prn) without an affinity tag using an Escherichia coli expression system. Compared to the native Prn (n-Prn) from B. pertussis, the recombinant protein showed a comparable reactivity in Western blotting and ELISA as well as a similar structure as analyzed by circular dichroism, fluorescence spectroscopy and size-exclusion chromatography. Furthermore, parenteral immunization of mice with native or r-Prn produced a similar increase in serum anti-Prn antibodies and similar protective activity following aerosol challenge. Our results indicate that the recombinant protein approach may be useful for developing a potential component of APVs as well as an antigen material which can be used in both clinical epidemiological evaluation and laboratory vaccine evaluation studies. Moreover, these studies also provide further evidence for the role of Prn in pertussis immunity.     

Characterization of the key antigenic components of pertussis vaccine based on outer membrane vesicles

Pertussis has resurged during the last two decades in different countries. In particular in the 2010–2013 period large outbreaks were detected in US, Australia, UK and The Netherlands with significant mortality in infants. The epidemiological situation of pertussis points out the need to develop new vaccines and in this regard we previously developed a new vaccine based on outer membrane vesicles (OMVs) which have been shown to be safe and to induce protection in mice. Here we have further investigated the properties of OMVs vaccines; in particular we studied the contribution of pertussis toxin (PTx) and pertactin (Prn) in OMVs-mediated protection against pertussis. PTx-deficient OMVs and Prn-deficient OMVs were obtained from defective Bordetella pertussis mutants. The absence of PTx or Prn did compromise the protective capacity of the OMVs formulated as Tdap vaccine. Whereas the protective efficacy of the PTx-deficient OMVs in mice was comparable to Prn-deficient OMVs, the protective capacity of both of them was significantly impaired when it was compared with the wild type OMVs. Interestingly, using OMVs obtained from a B. pertussis strain which does not express any of the virulence factors but expresses the avirulent phenotype; we observed that the protective ability of such OMVs was lower than that of OMVs obtained from virulent B. pertussis phase. However, it was surprising that although the protective capacity of avirulent OMVs was lower, they were still protective in the used mice model. These results allow us to hypothesize that OMVs from avirulent phase shares protective components with all OMVs assayed. Using an immune proteomic strategy we identified some common components that could play an important role in protection against pertussis.     

Connaissance et application des recommandations vaccinales concernant la coqueluche par la médecine du travail des établissements de santé de Paris

Objective

A questionnaire was used on 44 public and private hospital physicians in Paris to evaluate their knowledge of and adherence to Vaccination Guidelines, three years after their introduction.

Results

Eighty per cent of the physicians answered and 92.5% were aware of the vaccination guidelines but only 2 out of 4 respected the targeted vaccination in young adults even when the vaccine was available. A policy of pertussis vaccination was applied only in 12 institutions, but even in these, the rate of vaccinated healthcare workers remained low or was not documented.

Conclusion

Pertussis is a potential risk to newborns not or partially vaccinated in France. Even if the vaccine is available, adherence to pertussis vaccination guidelines must be improved. Efforts should be made to better publicize and apply pertussis vaccination guidelines.     

Adjuvanticity of native and detoxified adenylate cyclase toxin of Bordetella pertussis towards co-administered antigens

The cell-invasive adenylate cyclase toxin (CyaA) of Bordetella pertussis was shown to be highly antigenic in mice, stimulating serum anti-CyaA IgG antibody responses which were able to neutralise the cytotoxic effect of CyaA on J774.2 macrophage-like cells. The effect of co-administration to mice of the fully functional CyaA toxin or a toxin lacking adenylate cyclase enzymic activity (CyaA*) with other antigens from B. pertussis, namely pertussis toxin (PT) or pertussis toxoid (PTd), filamentous haemagglutinin (FHA) and pertactin (PRN), was investigated. CyaA* enhanced the serum IgG antibody responses to each of these antigens whereas, with CyaA, only anti-PRN antibody titres showed a modest increase. Peritoneal macrophages and spleen cells, collected at 2 weeks post-immunisation, were cultured and tested for nitric oxide (NO) and IFNgamma production, respectively, after stimulation in vitro with heat-killed B. pertussis cells or CyaA proteins. NO and IFNgamma production were higher in cells collected from mice immunised with CyaA or CyaA* in combination with a PT, FHA and PRN antigen mixture than from those taken from mice injected with antigen mixture alone, again with CyaA* acting as a better adjuvant than CyaA. The apparent enhancement of immune responses to the antigen mixture by CyaA* in particular was not paralleled by increased protection of mice against aerosol challenge with B. pertussis, but a statistically significant increase in protection was seen after intranasal challenge with B. parapertussis.