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.     

Intranasal murine model of Bordetella pertussis infection: II. Sequence variation and protection induced by a tricomponent acellular vaccine.

When pertussis toxin S1 subunit and pertactin structural genes in Bordetella pertussis clinical isolates from France and Germany were sequenced, 3 previously described S1 subunit types (S1 A, B and E), and 4 pertactin types (PRN A, B, C, A*) were found. PRN A*, present in the WHO reference strain 18323, was not described previously. In a respiratory mouse model, a tricomponent acellular pertussis vaccine (Infanrix) was highly effective in promoting lung clearance of all isolates expressing different S1 subunit and pertactin suggesting that use of acellular vaccine will not increase the risks of pertussis infection by these B. pertussis variants.     

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.     

Pertactin negative Bordetella pertussis demonstrates higher fitness under vaccine selection pressure in a mixed infection model

Whooping cough or pertussis is a highly infectious respiratory disease in humans caused by Bordetella pertussis. The use of acellular vaccines (ACV) has been associated with the recent resurgence of pertussis in developed countries including Australia despite high vaccination coverage where B. pertussis strains that do not express pertactin (Prn), a key antigenic component of the ACV, have emerged and become prevalent. In this study, we used an in vivo competition assay in mice immunised with ACV and in naïve (control) mice to compare the proportion of colonisation with recent clinical Prn positive and Prn negative B. pertussis strains from Australia. The Prn negative strain colonised the respiratory tract more effectively than the Prn positive strain in immunised mice, out-competing the Prn positive strain by day 3 of infection. However, in control mice, the Prn positive strain out-competed the Prn negative strain. Our findings of greater ability of Prn negative strains to colonise ACV-immunised mice are consistent with reports of selective advantage for these strains in ACV-immunised humans.     

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.     

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.     

Purification design and practice for pertactin,the third component of acellular pertussis vaccine,from Bordetella pertussis

Development of acellular pertussis vaccine (aPV) requires purification of several components from Bordetella pertussis. While the components pertussis toxin (PT) and filamentous hemagglutinin (FHA) have been successfully purified, the third component, pertactin, proves to be a difficult target due to its very low concentration. In order to solve its purification problem, we performed the surface potential analysis with GRASP2 program. The results demonstrated that there are two major charge patches, one negative and one positive, which are located separately on this linear protein. For this special feature, we designed a dual ion exchange chromatography strategy including an anionic exchange and a cationic exchange process for separation of pertactin from the heat extract of B. pertussis. The initial anionic exchange chromatography concentrated the product from 1.7% to 14.6%, with recovery of 80%. The second cationic exchange chromatography increased the purity to 33%, with recovery of 83%. The final purification was accomplished by hydrophobic interaction chromatography, yielding a purity of 96%. The total recovery of the three columns was 61%. Characterization of the purified antigen was performed with CD, intrinsic fluorescence, HP-SEC and western-blot, showing that the purified protein kept its natural conformation and immune-reactivity. The rationally designed process proved to be feasible, and it is suitable for large-scale preparation of the third aPV component pertactin.     

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.     

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.     

Construction and evaluation of a pertactin-deficient live attenuated pertussis vaccine candidate BPZE1 derivative

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.     

Pertactin-Deficient Bordetella pertussis with Unusual Mechanism of Pertactin Disruption,Spain, 1986–2018

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.     

A novel method for evaluating natural and vaccine induced serological responses to Bordetella pertussis antigens

We studied the time course of serum IgG antibodies against 3 different pertussis vaccine antigens: PT (pertussis toxin), FHA (filamentous hemagglutinin), Prn (pertactin) in sera from individuals vaccinated with four different pertussis vaccines at 4 years of age: (N = 44, 44, 23 and 23, respectively,) and compared the responses to/after natural infection with Bordetella pertussis (N = 44, age 1–8 years).     

B-cell responses after intranasal vaccination with the novel attenuated Bordetella pertussis vaccine strain BPZE1 in a randomized phase I clinical trial

Despite high vaccination coverage, pertussis is still a global concern in infant morbidity and mortality, and improved pertussis vaccines are needed. A live attenuated Bordetella pertussis strain, named BPZE1, was designed as an intranasal vaccine candidate and has recently been tested in man in a phase I clinical trial. Here, we report the evaluation of the B-cell responses after vaccination with BPZE1. Forty-eight healthy males with no previous pertussis-vaccination were randomized into one of three dose-escalating groups or into a placebo group. Plasma blast- and memory B-cell responses were evaluated by ELISpot against three different pertussis antigens: pertussis toxin, filamentous haemagglutinin and pertactin. Seven out of the 36 subjects who had received the vaccine were colonized by BPZE1, and significant increases in the memory B-cell response were detected against all three tested antigens in the culture-positive subjects between days 0 and 28 post-vaccination. The culture-positive subjects also mounted a significant increase in the filamentous haemagglutinin-specific plasma blast response between days 7 and 14 post-vaccination. No response could be detected in the culture-negatives or in the placebo group post-vaccination. These data show that BPZE1 is immunogenic in humans and is therefore a promising candidate for a novel pertussis vaccine. This trial is registered at ClinicalTrials.gov (NCT01188512).     

Needle-free and adjuvant-free epicutaneous boosting of pertussis immunity: Preclinical proof of concept

The limited durability of pertussis vaccine-induced protection requires novel approaches to reactivate immunity and limit pertussis resurgence in older children and adults. We propose that periodic boosters could be delivered using a novel epicutaneous delivery system (Viaskin^®) to deliver optimized pertussis antigens such as genetically-detoxified pertussis toxin (rPT). To best mimic the human situation in which vaccine-induced memory cells persist, whereas antibodies wane, we developed a novel adoptive transfer murine model of pertussis immunity. This allowed demonstrating that a single application of Viaskin^® delivering rPT and/or pertactin and filamentous hemagglutinin effectively reactivates vaccine-induced pertussis immunity and protects against Bordetella pertussis challenge. Recalling pertussis immunity without needles nor adjuvant may considerably facilitate the acceptance and application of periodic boosters.     

No evidence of cross-reactivity of human antibodies to a 33-mer peptide of the alpha-gliadin component of gluten with Bordetella pertussis pertactin

A 33-mer peptide of the alpha-gliadin component of gluten was recently identified as primary initiator of the inflammatory response to gluten in coeliac disease (CD) patients. This proline-glutamine-rich peptide (PG-peptide) is highly homologous to internal sequence of pertactin, an immunogenic protein of Bordetella pertussis. Using enzyme immunoassays, we measured serum antibodies to pertactin and to PG-peptide in 167 Finnish subjects including pertussis vaccine recipients and pertussis patients, CD and non-CD patients and healthy individuals. We found no cross-reactivity between human antibodies to the two different components, suggesting that neither pertussis immunization nor disease contributes to the pathogenesis of CD.     

A search for serologic correlates of immunity to Bordetella pertussis cough illnesses

In a pertussis vaccine efficacy trial in Germany we collected sera from vaccinees (DTaP or DTP) after the third and fourth doses of vaccine or at comparable time periods in DT vaccine recipients. In addition, sera were collected from a randomized sample of subjects in each vaccine group at approximately 3-month intervals from which antibody kinetic curves were constructed, which allowed us to estimate specific antibody values to pertussis toxin (PT), filamentous hemagglutinin (FHA), pertactin and fimbriae-2 at the time of exposure in the household setting. The imputed geometric mean antibody values to PT, pertactin and fimbriae-2 at the time of household exposure to Bordetella pertussis infection were higher (p < 0.07 or lower) in non-cases compared with cases. A multivariate (classification tree) analysis found that only pertactin and PT were significant in protection. Subjects with an imputed pertactin value of < 7 EU ml⁻¹ had a 67% (18/27) chance of infection regardless of the PT value. If the pertactin value was ≥ 7 EU ml⁻¹ and the PT value ≥ 66 EU ml⁻¹ all subjects were non-cases. If the pertactin value was ≥ 7 and the PT value was < 66 EU ml⁻¹ the predicted probability of being a case was 31% (15/49). Logistic regression analysis also found that high versus low pertactin values were associated with illness prevention following household exposure. In the presence of antibody to pertactin, PT and fimbriae-2, the additional presence of antibody to FHA did not contribute to protection. Our data support historical data indicating that agglutinating antibodies are associated with protection and also recent serologic correlates data and clinical efficacy data which indicate that multicomponent vaccines containing pertactin and fimbriae have better efficacy than PT or PT/FHA vaccines.     

Appearance of Fim3 and ptxP3-Bordetella pertussis strains, in two regions of Sweden with different vaccination programs

After introduction of a mono-component vaccine, containing only pertussis toxoid (PT), the incidence of pertussis was significantly higher in the Gothenburg area among children during the period October 1, 1997 until end of 2006 compared to the Rest of Sweden where a vaccine containing PT and two other pertussis antigens was used. To investigate a possible cause of this difference, the Bordetella pertussis populations in both regions were compared by determining the fimbrial serotype (Fim), the PFGE-type and the pertussis toxin promoter allele type (ptxP). Strains with the ptxP1 allele were successively replaced by ptxP3 strains producing more pertussis toxin. In Gothenburg compared to the Rest of Sweden, Fim3 and ptxP3 strains were observed earlier and reached higher frequencies in the studied period. Since ptxP3 strains have been shown to be more virulent, their higher prevalence may have contributed to the higher incidence of pertussis in the Gothenburg area. In addition we found a high degree of linkage between PFGE-profile and ptxP3. Our results highlight the importance of strain typing to gain insight into the mechanisms of immunity-associated selection of microbial subtypes and the causes of changes in incidences of infectious diseases.     

Immunogenicity of the reduced-antigen-content dTpa vaccine (Boostrix(®)) in adults 55 years of age and over: a sub-analysis of four trials

Background

Older adults, especially those over 65 years, are at risk of more severe morbidity from diphtheria, tetanus and pertussis and may transmit pertussis to unvaccinated or not yet fully vaccinated infants, but data on their response to reduced-antigen-content tetanus, diphtheria and acellular pertussis (dTpa) vaccines are lacking.

Methods

A sub-analysis pooled immunogenicity results in 293 adults aged 55+ years (mean age 64.4 years) from four randomised, controlled clinical trials of dTpa vaccine (Boostrix^®, GlaxoSmithKline Biologicals) with or without IPV co-administration, or dTpa-IPV (Boostrix^® IPV).

Results

Seroprotective antibody levels were achieved by 82.8% for diphtheria and 94.5% for tetanus. For pertussis antigens, the booster response rate, defined as initially seronegative subjects [<5 EU/mL] reaching ≥5 EU/mL; or a ≥2-fold increase in antibody concentration if initially seropositive was 89.2% for pertussis toxoid, 95.8% for filamentous haemagglutinin and 94.5% for pertactin. Post-booster geometric mean concentrations (GMC) increased for all antigens. Post-booster anti-tetanus and anti-PRN GMCs tended to be higher in 55- to 64-year olds than in those aged 65+.

Conclusion

Larger numbers of subjects over 75 years are needed to better define responses in advanced age, but these data suggest that a single booster dose of dTpa or dTpa-IPV induces good immunological responses in most, and that these vaccines could be readily integrated into existing programmes.     

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.     

Different IgG-subclass distributions after whole-cell and acellular pertussis infant primary vaccinations in healthy and pertussis infected children

The distribution of IgG-subclasses provides insight in the immunological mechanisms of protection against whooping cough. We investigated the effect of Dutch whole-cell pertussis and acellular pertussis vaccines administered in infancy on the IgG-subclass distributions in healthy children aged 12 months, 4 years and 9 years as well as in children who have been infected with Bordetella pertussis.A fluorescent bead-based multiplex immunoassay was used for the measurement of IgG1, IgG2, IgG3 and IgG4 responses against pertussis toxin, filamentous heamagglutinin and pertactin.Although IgG1 was the predominant subclass for all pertussis antigens in both healthy and infected children, elevated IgG4 levels were only present in children who had received repeated number of acellular pertussis vaccinations. IgG2 and IgG3 antibodies did not contribute to the IgG response. No differences in IgG-subclasses between healthy vaccinated or infected children were found.The pertussis vaccine used for priming seems to determine the IgG-subclass composition elicited after a secondary antibody response either induced by pertussis vaccination or infection. The pronounced anti-pertussis IgG4 response might reflect the Th2-skewing of the immune response after aP vaccination.