Bordetella pertussis and vaccination: The persistence of a genetically monomorphic pathogen

Before childhood vaccination was introduced in the 1950s, pertussis or whooping cough was a major cause of infant death worldwide. Widespread vaccination of children was successful in significantly reducing morbidity and mortality. However, despite vaccination, pertussis has persisted and, in the 1990s, resurged in a number of countries with highly vaccinated populations. Indeed, pertussis has become the most prevalent vaccine-preventable disease in developed countries with estimated infection frequencies of 1–6%. Recently vaccinated children are well protected against pertussis disease and its increase is mainly seen in adolescents and adults in which disease symptoms are often mild. The etiologic agent of pertussis, Bordetella pertussis, is extremely monomorphic and its ability to persist in the face of intensive vaccination is intriguing. Numerous studies have shown that B. pertussis populations changed after the introduction of vaccination suggesting adaptation. These adaptations did not involve the acquisition of novel genes but small genetic changes, mainly SNPs, and occurred in successive steps in a period of 40 years. The earliest adaptations resulted in antigenic divergence with vaccine strains. More recently, strains emerged with increased pertussis toxin (Ptx) production. Here I argue that the resurgence of pertussis is the compound effect of pathogen adaptation and waning immunity. I propose that the removal by vaccination of naïve infants as the major source for transmission was the crucial event which has driven the changes in B. pertussis populations. This has selected for strains which are more efficiently transmitted by primed hosts in which immunity has waned. The adaptation of B. pertussis to primed hosts involved delaying an effective immune response by antigenic divergence with vaccine strains and by increasing immune suppression through higher levels of Ptx production. Higher levels of Ptx may also benefit transmission by enhancing clinical symptoms. The study of B. pertussis populations has not only increased our understanding of pathogen evolution, but also suggests way to improve pertussis vaccines, underlining the public health significance of population-based studies of pathogens.     

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 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.     

Genetic diversity and population dynamics of Bordetella pertussis in China between 1950–2007

Pertussis is an acute respiratory infectious disease caused by the bacterium Bordetella pertussis. Although pertussis vaccination was introduced in the 1960s, pertussis is still an endemic disease in China. To better understand the genetic diversity of the Chinese B. pertussis population, we characterized 115 clinical isolates obtained in China during 1950–2007 using multilocus variable-number tandem repeat analysis (MLVA). Forty-six different B. pertussis MLVA profiles (MTs) were identified, of which 13 were new MTs. Analysis using a minimum-spanning tree showed that distinct MTs were prevalent during different periods, suggesting that a dynamic change in B. pertussis MTs occurred over time in China. The predominant MTs in recent isolates from China were different from those of many developed countries. A decreasing trend in genetic diversity of the B. pertussis population was observed following the introduction of pertussis vaccines. Similar to the pertactin 2 (prn2) allele, the novel pertussis toxin promoter (ptxP3) allele first emerged in 2000, but unlike trends elsewhere, ptxP1 remained predominant among the isolates, further reflecting the unique temporal trends in the B. pertussis population in China. Our results suggest that temporal changes in the B. pertussis population may be closely associated with vaccination coverage and the vaccine types used. These data may lead to an improved understanding of the virulence mechanism of B. pertussis and facilitate new strategies for controlling this infectious disease.     

Long-term immunity against pertussis induced by a single nasal administration of live attenuated B. pertussis BPZE1

Duration of vaccine-induced immunity plays a key role in the epidemiology and in the pattern of transmission of a vaccine-preventable disease. In the case of whooping cough, its re-emergence has been attributed, at least partly, to the waning of immunity conferred by current pertussis vaccines. We have recently developed a highly attenuated live vaccine, named BPZE1, which has been shown to be safe and to induce strong protective immunity against Bordetella pertussis infection in mice. In this study, we evaluated the long-term immunogenicity and protective efficacy induced by a single intranasal dose of BPZE1. Up to 1 year after immunization, BPZE1 showed significantly higher efficacy to protect adult and infant mice against B. pertussis infection than two administrations of an acellular pertussis vaccine (aPV). B. pertussis-specific antibodies were induced by live BPZE1 and by aPV, with increasing amounts during the first 6 months post-immunization before a progressive decline. Cell-mediated immunity was also measured 1 year after immunization and showed the presence of memory T cells in the spleen of BPZE1-immunized mice. Both cell-mediated and humoral immune responses were involved in the long-lasting protection induced by BPZE1, as demonstrated by adoptive transfer experiments to SCID mice. These data highlight the potential of the live attenuated BPZE1 candidate vaccine as part of a strategy to solve the problem of waning protective immunity against B. pertussis observed with the current aPV vaccines.     

Construction and evaluation of Bordetella pertussis live attenuated vaccine strain BPZE1 producing Fim3

Pertussis or whooping cough is currently the most prevalent vaccine-preventable childhood disease despite >85% global vaccination coverage. In recent years incidence has greatly increased in several high-income countries that have switched from the first-generation, whole-cell vaccine to the newer acellular vaccines, calling for improved vaccination strategies with better vaccines. We have developed a live attenuated pertussis vaccine candidate, called BPZE1, which is currently in clinical development. Unlike other pertussis vaccines, BPZE1 has been shown to provide strong protection against infection by the causative agent of pertussis, Bordetella pertussis, in non-human primates. BPZE1 is a derivative of the B. pertussis strain Tohama I, which produces serotype 2 (Fim2) but not serotype 3 fimbriae (Fim3). As immune responses to fimbriae are likely to contribute to protection, we constructed a BPZE1 derivative, called BPZE1f3, that produces both serotypes of fimbriae. Whereas nasal vaccination of mice with BPZE1 induced antibodies to Fim2 but not to Fim3, vaccination with BPZE1f3 elicited antibodies to both Fim2 and Fim3 at approximately the same level. In mice, both BPZE1 and BPZE1f3 provided equal levels of protection against clinical isolates that either produce Fim2 alone, both Fim2 and Fim3, or no fimbriae. However, vaccination with BPZE1f3 provided significantly stronger protection against Fim3-only producing B. pertussis than vaccination with BPZE1, indicating that immune responses to fimbriae contribute to serotype-specific protection against B. pertussis infection.     

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.     

Rapid Increase in Pertactin-deficient Bordetella pertussis Isolates,Australia

Acellular vaccines against Bordetella pertussis were introduced in Australia in 1997. By 2000, these vaccines had replaced whole-cell vaccines. During 2008–2012, a large outbreak of pertussis occurred. During this period, 30% (96/320) of B. pertussis isolates did not express the vaccine antigen pertactin (prn). Multiple mechanisms of prn inactivation were documented, including IS481 and IS1002 disruptions, a variation within a homopolymeric tract, and deletion of the prn gene. The mechanism of lack of expression of prn in 16 (17%) isolates could not be determined at the sequence level. These findings suggest that B. pertussis not expressing prn arose independently multiple times since 2008, rather than by expansion of a single prn-negative clone. All but 1 isolate had ptxA1, prn2, and ptxP3, the alleles representative of currently circulating strains in Australia. This pattern is consistent with continuing evolution of B. pertussis in response to vaccine selection pressure.     

Reciprocal interference of maternal and infant immunization in protection against pertussis

BackgroundBecause of the current re-emergence of pertussis, vaccination during the 3rd trimester of pregnancy is recommended in several countries in order to protect neonates by placental transfer of maternal antibodies. Here, we examined the potential reciprocal interference of mother and infant vaccination in protection against pertussis in mice.MethodsFemale mice were vaccinated with acellular pertussis vaccines and protection against Bordetella pertussis challenge, as well as functional antibodies were measured in their offspring with or without re-vaccination.ResultsMaternal immunization protected the offspring against B. pertussis challenge, but protection waned quickly and was lost after vaccination of the infant mice with the same vaccine. Without affecting antibody titers, infant vaccination reduced the protective functions of maternally-derived antibodies, evidenced both in vitro and in vivo. Protection induced by infant vaccination was also affected by maternal antibodies. However, when mothers and infants were immunized with two different vaccines, no interference of infant vaccination on the protective effects of maternal antibodies was noted.ConclusionIt may be important to determine the functionality of antibodies to evaluate potential interference of maternal and infant vaccination 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.     

Bordetella pertussis Strains with Increased Toxin Production Associated with Pertussis Resurgence

Before childhood vaccination was introduced in the 1940s, pertussis was a major cause of infant death worldwide. Widespread vaccination of children succeeded in reducing illness and death. In the 1990s, a resurgence of pertussis was observed in a number of countries with highly vaccinated populations, and pertussis has become the most prevalent vaccine-preventable disease in industrialized countries. We present evidence that in the Netherlands the dramatic increase in pertussis is temporally associated with the emergence of Bordetella pertussis strains carrying a novel allele for the pertussis toxin promoter, which confers increased pertussis toxin (Ptx) production. Epidemiologic data suggest that these strains are more virulent in humans. We discuss changes in the ecology of B. pertussis that may have driven this adaptation. Our results underline the importance of Ptx in transmission, suggest that vaccination may select for increased virulence, and indicate ways to control pertussis more effectively.     

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.     

Characterization of the immune response induced by pertussis OMVs-based vaccine

For the development of a third generation of pertussis vaccine that could improve the control of the disease, it was proposed that the immune responses induced by the classic whole cell vaccine (wP) or after infection should be used as a reference point. We have recently identified a vaccine candidate based on outer membrane vesicles (OMVs) derived from the disease etiologic agent that have been shown to be safe and protective in mice model of infection. Here we characterized OMVs-mediated immunity and the safety of our new candidate. We also deepen the knowledge of the induced humoral response contribution in pertussis protection. Regarding the safety of the OMVs based vaccine (Tdap_OMVsBp,) the in vitro whole blood human assay here performed, showed that the low toxicity of OMVs-based vaccine previously detected in mice could be extended to human samples.Stimulation of splenocytes from immunized mice evidenced the presence of IFN-γ and IL-17-producing cells, indicated that OMVs induces both Th1 and Th17 response. Interestingly Tdap_OMVsBp-raised antibodies such as those induced by wP and commercial acellular vaccines (aP) which contribute to induce protection against Bordetella pertussis infection. As occurs with wP-induced antibodies, the TdapOMVsBp-induced serum antibodies efficiently opsonized B. pertussis. All the data here obtained shows that OMVs based vaccine is able to induce Th1/Th17 and Th2 mixed profile with robust humoral response involved in protection, positioning this candidate among the different possibilities to constitute the third generation of anti-pertussis vaccines.     

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.     

Immune response to Bordetella pertussis is associated with season and undernutrition in Senegalese children

BackgroundWhile vaccines elicit a protective response in most recipients, studies suggest that environmental and nutritional factors can influence the strength of the individual response to immunization and to subsequent natural infectious challenges.MethodsWe conducted a longitudinal survey in Senegal to assess the individual response to B. pertussis, a respiratory disease against which Senegalese children are vaccinated before the age of one (Clinicaltrials.gov ID: NCT01545115). A cohort of 203 children aged 1–9 from four villages of the Senegal River Valley was followed-up for 14 months (October 2008–January 2010). During that period, four visits have been made to the villages to assess the immunological and nutritional status of these children and to determine risk factors involved in the modulation of their humoral immune response to B. pertussis toxin.ResultsA multivariate model has demonstrated that birth season and nutritional status appeared to modulate humoral response to pertussis toxin. Moreover, response to B. pertussis was dependent on age, village and time of visit.ConclusionsThese results are consistent with the hypothesis that environmental and nutritional factors modulate children's response to pertussis following natural infection or vaccination.     

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.     

Pertactin-Deficient Bordetella pertussis,Vaccine-Driven Evolution,and Reemergence of Pertussis

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

CpG 1018® adjuvant enhances Tdap immune responses against Bordetella pertussis in mice

Bordetella pertussis is the causative agent of whooping cough (pertussis), a severe respiratory disease that can be fatal, particularly in infants. Despite high vaccine coverage, pertussis remains a problem because the currently used DTaP and Tdap vaccines do not completely prevent infection or transmission. It is well established that the alum adjuvant is a potential weakness of the acellular vaccines because the immunity provided by it is short-term. We aimed to evaluate the potential of CpG 1018® adjuvant to improve antibody responses and enhance protection against B. pertussis challenge in a murine model. A titrated range of Tdap vaccine doses were evaluated in order to best identify the adjuvant capability of CpG 1018. Antibody responses to pertussis toxin (PT), filamentous hemagglutinin (FHA), or the whole bacterium were increased due to the inclusion of CpG 1018. In B. pertussis intranasal challenge studies, we observed improved protection and bacterial clearance from the lower respiratory tract due to adding CpG 1018 to 1/20th the human dose of Tdap. Further, we determined that Tdap and Tdap + CpG 1018 were both capable of facilitating clearance of strains that do not express pertactin (PRN^-), which are rising in prevalence. Functional phenotyping of antibodies revealed that the inclusion of CpG 1018 induced more bacterial opsonization and antibodies of the Th1 phenotype (IgG2a and IgG2b). This study demonstrates the potential of adding CpG 1018 to Tdap to improve immunogenicity and protection against B. pertussis compared to the conventional, alum-only adjuvanted Tdap vaccine.     

Will we have new pertussis vaccines?

Despite wide vaccination coverage with efficacious vaccines, pertussis is still not under control in any country. Two types of vaccines are available for the primary vaccination series, diphtheria/tetanus/whole-cell pertussis and diphtheria/tetanus/acellular pertussis vaccines, in addition to reduced antigen content vaccines recommended for booster vaccination. Using these vaccines, several strategies are being explored to counter the current pertussis problems, including repeated vaccination, cocoon vaccination and maternal immunization. With the exception of the latter, none have proven their effectiveness, and even maternal vaccination is not expected to ultimately control pertussis. Therefore, new pertussis vaccines are needed, and several candidates are in early pre-clinical development. They include whole-cell vaccines with low endotoxin content, outer membrane vesicles, new formulations, acellular vaccines with new adjuvants or additional antigens and live attenuated vaccines. The most advanced is the live attenuated nasal vaccine BPZE1. It provides strong protection in mice and non-human primates, is safe, even in immune compromised animals, and genetically stable after in vitro and in vivo passages. It also has interesting immunoregulatory properties without being immunosuppressive. It has successfully completed a first-in-man clinical trial, where it was found to be safe, able to transiently colonize the human respiratory tract and to induce immune responses in the colonized subjects. It is now undergoing further clinical development. As it is designed to reduce carriage and transmission of Bordetella pertussis, it may hopefully contribute to the ultimate control of pertussis.     

Modeling Immune Evasion and Vaccine Limitations by Targeted Nasopharyngeal Bordetella pertussis Inoculation in Mice

Conventional pertussis animal models deliver hundreds of thousands of Bordetella pertussis bacteria deep into the lungs, rapidly inducing severe pneumonic pathology and a robust immune response. However, human infections usually begin with colonization and growth in the upper respiratory tract. We inoculated only the nasopharynx of mice to explore the course of infection in a more natural exposure model. Nasopharyngeal colonization resulted in robust growth in the upper respiratory tract but elicited little immune response, enabling prolonged and persistent infection. Immunization with human acellular pertussis vaccine, which prevents severe lung infections in the conventional pneumonic infection model, had little effect on nasopharyngeal colonization. Our infection model revealed that B. pertussis can efficiently colonize the mouse nasopharynx, grow and spread within and between respiratory organs, evade robust host immunity, and persist for months. This experimental approach can measure aspects of the infection processes not observed in the conventional pneumonic infection model.