Audrey J. King

Multilocus Sequence Typing of Bordetella pertussis Based on Surface Protein Genes

ABSTRACT Despite more than 50 years of vaccination, Bordetella pertussis has remained endemic in The Netherlands, causing epidemic outbreaks every 3 to 5 years. Strain variation may play a role in the persistence of B. pertussis and was studied by sequencing 15 genes coding for surface proteins, including genes for all five components of acellular pertussis vaccines: pertussis toxin (Ptx), pertactin (Prn), filamentous hemagglutinin, and fimbriae (Fim2 and Fim3). A low level of allelic variation was observed, confirming a recent evolutionary origin of B. pertussis. In modern isolates, polymorphism was observed only in prn, ptxS1, ptxS3, and tcfA. Polymorphism in ptxS1, ptxS3, and tcfA was used to categorize isolates in multilocus sequence types (MLSTs). Analysis of Dutch isolates from 1949 to 1999 revealed five MLSTs, which showed a highly dynamic temporal behavior. We observed significant changes in the MLSTs after the introduction of pertussis vaccination in The Netherlands. Epidemic years were found to be associated with the expansion of MLST-4 or MLST-5. MLST-5 showed a remarkable expansion from 10% in 1997 to 80% in 1999. The MLST analysis was extended to a number of widely separated geographic regions: Finland, Italy, Japan, and the United States. MLST-4 and MLST-5 were found to dominate in Italy and the United States. In Finland and Japan, MLST-3 and MLST-2, respectively, were predominant. Thus, although each region showed distinctive MLST frequencies, in three of the five regions MLST-4 and MLST-5 were predominant. These types may represent newly emerged, successful clones. The identification of highly successful clones may shed light on the question of how B. pertussis is able to maintain itself in vaccinated populations.

Role of the polymorphic region 1 of the Bordetella pertussis protein pertactin in immunity

In several countries pertussis is re-emerging, despite a high vaccination coverage. It is suggested that antigenic divergence between Bordetella pertussis vaccine strains and circulating strains, in particular with respect to pertactin, has contributed to pertussis re-emergence. Polymorphism in pertactin is essentially limited to region 1, which is composed of repeats and is located adjacent to an Arg-Gly-Asp motif implicated in adherence. Evidence is provided for the immunological relevance of polymorphism in region 1. Region 1 was found to contain a B-cell epitope recognized in both humans and mice. Furthermore, variation in region 1 affected antibody binding and, in a mouse respiratory infection model, the efficacy of a whole-cell vaccine. Moreover, passive and active immunization indicated that region 1 confers protective immunity. An mAb directed against a linear conserved epitope conferred cross-immunity against isolates with distinct pertactin variants. The results indicate an important role of region 1 of pertactin in immunity.

Changes in the genomic content of circulating Bordetella pertussis strains isolated from the Netherlands, Sweden, Japan and Australia: adaptive evolution or drift?

BackgroundBordetella pertussis is the causative agent of human whooping cough (pertussis) and is particularly severe in infants. Despite worldwide vaccinations, whooping cough remains a public health problem. A significant increase in the incidence of whooping cough has been observed in many countries since the 1990s. Several reasons for the re-emergence of this highly contagious disease have been suggested. A particularly intriguing possibility is based on evidence indicating that pathogen adaptation may play a role in this process. In an attempt to gain insight into the genomic make-up of B. pertussis over the last 60 years, we used an oligonucleotide DNA microarray to compare the genomic contents of a collection of 171 strains of B. pertussis isolates from different countries.ResultsThe CGH microarray analysis estimated the core genome of B. pertussis, to consist of 3,281 CDSs that are conserved among all B. pertussis strains, and represent 84.8% of all CDSs found in the 171 B. pertussis strains. A total of 64 regions of difference consisting of one or more contiguous CDSs were identified among the variable genes. CGH data also revealed that the genome size of B. pertussis strains is decreasing progressively over the past 60 years. Phylogenetic analysis of microarray data generated a minimum spanning tree that depicted the phylogenetic structure of the strains. B. pertussis strains with the same gene content were found in several different countries. However, geographic specificity of the B. pertussis strains was not observed. The gene content was determined to highly correlate with the ptxP-type of the strains.ConclusionsAn overview of genomic contents of a large collection of isolates from different countries allowed us to derive a core genome and a phylogenetic structure of B. pertussis. Our results show that B. pertussis is a dynamic organism that continues to evolve.

Epitope Structure of the Bordetella pertussis Protein P.69 Pertactin, a Major Vaccine Component and Protective Antigen

ABSTRACT Bordetella pertussis is reemerging in several countries with a traditionally high vaccine uptake. An analysis of clinical isolates revealed antigenic divergence between vaccine strains and circulating strains with respect to P.69 pertactin. Polymorphisms in P.69 pertactin are mainly limited to regions comprised of amino acid repeats, designated region 1 and region 2. Region 1 flanks the RGD motif, which is involved in adherence. Although antibodies against P.69 pertactin are implicated in protective immunity, little is known about the structure and location of its epitopes. Here we describe the identification by pepscan analysis of the locations of mainly linear epitopes recognized by human sera and mouse monoclonal antibodies (MAbs). A total of 24 epitopes were identified, and of these only 2 were recognized by both MAbs and human antibodies in serum. A number of immunodominant epitopes were identified which were recognized by 78 to 93% of the human sera tested. Blocking experiments indicated the presence of high-avidity human antibodies against conformational epitopes. Human antibodies against linear epitopes had much lower avidities, as they were unable to block MAbs. Pepscan analyses revealed several MAbs which bound to both region 1 and region 2. The two regions are separated by 289 amino acids in the primary structure, and we discuss the possibility that they form a single conformational epitope. Thus, both repeat regions may serve to deflect the immune response targeted to the functional domain of P.69 pertactin. This may explain why the variation in P.69 pertactin is so effective, despite the fact that it is limited to only two small segments of the molecule.

Characterization of Bordetella holmesii isolates from patients with pertussis-like illness in the Netherlands

Bordetella holmesii is a recently described human pathogen mainly isolated from blood. However, in the US and Canada, B. holmesii has also been cultured from the nasopharynx of patients with pertussis-like symptoms. To the best of our knowledge, respiratory isolates from Europe have not been characterized. Here, we report the isolation and characterization of B. holmesii from Dutch patients with pertussis-like illness. Species determination was confirmed by 16S rRNA gene sequencing and detection by PCR of IS481 and bhoE, a gene not found in Bordetella pertussis but present in B. holmesii. Comparative genomic hybridization (CGH) with microarrays revealed that the Dutch isolates formed a cluster distinct from isolates from the US and UK suggesting a distinct population or an epidemiological relationship between the Dutch isolates. All isolates contained a locus involved in iron uptake, previously suggested to originate from B. pertussis. The causes for the apparent increase in the isolation of B. holmesii are discussed.

Immunodominance in mouse and human CD4+ T-cell responses specific for the bordetella pertussis virulence factor P.69 pertactin

ABSTRACT P.69 pertactin (P.69 Prn), an adhesion molecule from the causative agent of pertussis, Bordetella pertussis, is present in cellular and most acellular vaccines that are currently used worldwide. Although both humoral immunity and cellular immunity directed against P.69 Prn have been implicated in protective immune mechanisms, the identities of CD4+ T-cell epitopes on the P.69 Prn protein remain unknown. Here, a single I-Ad-restricted B. pertussis conserved CD4+ T-cell epitope at the N terminus of P.69 Prn was identified by using a BALB/c T-cell hybridoma. The epitope appeared immunodominant among four other minor strain-conserved P.69 Prn epitopes recognized after vaccination and B. pertussis infection, and it was capable of evoking a Th1/Th17-type cytokine response. B. pertussis P.69 Prn immune splenocytes did not cross-react with natural variants of the epitope as present in Bordetella parapertussis and Bordetella bronchiseptica. Finally, it was found that the immunodominant P.69 Prn epitope is broadly recognized in the human population by CD4+ T cells in an HLA-DQ-restricted manner. During B. pertussis infection, the epitope was associated with a Th1-type CD4+ T-cell response. Hence, this novel P.69 Prn epitope is involved in CD4+ T-cell immunity after B. pertussis vaccination and infection in mice and, more importantly, in humans. Thus, it may provide a useful tool for the evaluation of the type, magnitude, and maintenance of B. pertussis-specific CD4+ T-cell mechanisms in preclinical and clinical vaccine studies.

Molecular typing of Bordetella parapertussis isolates circulating in Pakistan

Although a whole-cell pertussis vaccine was introduced in Pakistan in 1980, little is known about the pertussis prevalence and circulating strains in Pakistan. The aim of this study was to analyze Bordetella parapertussis isolates circulating between 2005 and 2009 in Pakistan and to compare them with those found in other countries during different periods. A total of 59 (7.35%) B.xa0parapertussis isolates from 802 subjects (median age, 3xa0years) from Pakistan, with pertussis-like symptoms were investigated. We carried out genotyping and DNA microarray analyses on these isolates and compared them with some international isolates of B.xa0parapertussis. We found that the allele for pertactin (prn) found in strains studied from Pakistan was identical to the predominant type found in Europe. We showed that B.xa0parapertussis isolates circulating in Pakistan are part of the same pulsed-field gel electrophoresis group to those circulating in Finland during the period of 1982-2007. Finally, microarray analysis confirmed that the isolates collected in Pakistan, were quite similar to international strains. Overall, these results confirm that B.xa0parapertussis is extremely monomorphic. The high isolation rate of B.xa0parapertussis (7.35%) compared to Bordetella pertussis (0.5%) may suggest that the whole-cell vaccine used in Pakistan is effective against B.xa0pertussis (0.5% infections detected), but much less so against B.xa0parapertussis.