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

Vaccine 27 (2009) 6034–6041

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First report and detailed characterization of B. pertussis isolates not expressing pertussis toxin or pertactin V. Bouchez a , D. Brun a , T. Cantinelli b , G. Dore a , E. Njamkepo a , N. Guiso a,∗ a b

Institut Pasteur, Unité Prévention et Thérapie Moléculaires des Maladies Humaines, URA-CNRS 3012, 25 rue du Dr Roux, 75015 Paris, France Institut Pasteur, Groupe à 5 ans Microorganismes et Barrières de l’hôte, Paris, France

a r t i c l e

i n f o

Article history: Received 16 March 2009 Received in revised form 16 June 2009 Accepted 22 July 2009 Available online 8 August 2009 Keywords: Bordetella pertussis Pertussis toxin Pertactin

a b s t r a c t 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. © 2009 Elsevier Ltd. All rights reserved.

1. Introduction The introduction of Pertussis vaccination in young children has dramatically decreased the incidence of the disease. However, it is still endemic in all regions where vaccination was introduced. Furthermore, vaccination of young children has modified the transmission of the disease. Before the introduction of vaccination, children were infected when entering collectivities at around 6–7 years of age and were the main reservoir of Bordetella pertussis, the bacterium responsible for the disease. After 50 years of intensive vaccination in young children, it became evident that adults and adolescents, whose immunity has waned with time in the absence of vaccinal (but also natural) boosters, became susceptible for the disease. They were shown to transmit the disease to vulnerable newborns too young to be vaccinated and for whom the consequences of the illness can be dramatic [1–3]. For this reason, boosters have been implemented in France in 1998 for adolescents, in 2004 for young parents and health care workers in contact with newborns, and in 2008 for all health care workers, for young adults 26–28 years of age, and for all adults who did not receive any pertussis booster [4]. Pertussis whole cell (Pw) vaccine was used between 1959 and 1998 for primo-vaccination and the first booster at 16–18 months. In 1998, pertussis acellular (Pa) vaccines were introduced in France for adolescent booster. Since 2002, the majority of young children are vaccinated with Pa vaccines. Two types of Pa vaccines are used for children and adolescents.

∗ Corresponding author. Tel.: +33 1 45 68 83 34; fax: +33 1 40 61 35 33. E-mail address: [email protected] (N. Guiso). 0264-410X/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2009.07.074

The one contains two pertussis antigens, namely Pertussis Toxin (PT) and filamentous hemagglutinin (FHA), and the other contains three pertussis antigens, PT, FHA and pertactin (PRN). In 2004, two more pertussis vaccines were introduced, for booster immunization of adults, one containing three antigens (PT, FHA and PRN) and the other five antigens (PT, FHA, PRN and two fimbrial proteins: FIM2 and FIM3). The surveillance of the disease in France, starting in 1996, is hospital-based (called RENACOQ), coordinated by the Ministry of Health. The result of a 10-year period shows an increase in the age of the presumed source of contamination. The proportion of parents identified as transmitter is increasing [5]. These data were confirmed by a recent transmission study [2]. The protective effect of increasing the number of Pa vaccine doses against severe pertussis in infants was also highlighted [6]. Thus, the expected consequence of increasing the number of vaccine boosters would be to reduce transmission to newborns and control the disease. However, another effect can be expected at the level of the pathogen itself. We previously performed a temporal analysis of the population of B. pertussis [7,8], in which we observed that the polymorphism of the population is very low, with the currently circulating isolates being different from the vaccine strains. This evolution was also observed in all regions of the world where infants and young children were intensively vaccinated [9,10] but not in a region of the world with low Pw vaccine coverage where isolates, similar to those circulating during the prevaccine era, are still circulating [11]. These studies suggest that Pw vaccines, targeting the whole bacterium, succeeded in controlling vaccine-related strains. However, B. pertussis isolates still circulate. These circulating isolates harbor less genetic material, a high number of insertion sequences and repeated sequences but still possess

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Table 1 Characteristics of the isolates described in the study. Isolates

Expression factor deficiency

Age of the patient in months

Vaccine status of the patient

Hospitalization status of the patient

Clinical symptoms

FR3749

PT (deletion of ptx operon)

3

Non-vaccinated

Yes

FR3693 FR3705 FR3708 FR3793

PRN (insertion of an IS481 in the PRN gene) PRN (insertion of an IS481 in the PRN gene) PRN (insertion of an IS481 in the PRN gene) PRN (deletion of a part of the PRN gene)

3 5 5 2.5

1 dose of Pa vaccine 1 dose of Pa vaccine Non-vaccinated Non-vaccinated

No Yes Yes Yes

Suspicion of pertussis but no hyperlymphocytosis Suspicion of pertussis Suspicion of pertussis Suspicion of pertussis Suspicion of pertussis

all the genes encoding the proteins implicated in virulence. Since 1998, Pa vaccines have been replacing Pw vaccines in France. This type of vaccines, containing only a few bacterial proteins, is targeting directly and exclusively the virulence of the bacterium. We recently hypothesized that the intensive use of Pa vaccines will eventually favor the circulation of isolates not or less expressing the vaccine antigens and may be also over-expressing other antigens [9]. According to this hypothesis, clinical symptoms could be modified and biological diagnoses, such as the one based on the detection of antibodies against vaccine antigens, will lead to underreporting. For this reason, surveillance of the disease is of extreme importance. One of the best ways to diagnose the disease in infants as well as in adults is hospital-based surveillance. Such surveillance is established in France since 1996 in order to analyze the impact of adolescent and adult boosters not only on the morbidity of the newborns population but also on the circulating isolates. In the present study, we analyzed the isolates collected by the National Centre of Surveillance and we describe, for the first time, PRN- and PTdeficient B. pertussis isolates that were collected in 2007 in France. Two of these isolates were analyzed by 454 GS-FLX pyrosequencing to characterize their genetic specificity. Pathogenicity was evaluated using cellular and animal models. 2. Materials and methods 2.1. Clinical isolates used in this study The five clinical isolates analyzed in this study are described in Table 1. They were characterized using classical bacteriological techniques as growth on Bordet Gengou agar containing sheep blood, API galeries, oxydase and urease tests, detection of brownish pigment and use of specific Bordetella antiserums. The expression of virulence antigens as well as genotyping of their structural genes were performed as detailed below. 2.2. Bacterial growth and DNA extraction B. pertussis isolates were grown at 36 ◦ C for 72 h on Bordet Gengou agar (BGA) supplemented with 15% defibrinated sheep blood, and sub-cultured 24 h in the same medium before use. For 454 pyrosequencing, genomic DNA (gDNA) was prepared by using the Genomic-tip 500/G anion-exchange columns (Qiagen), according to the manufacturer’s recommendation. For PCR validation, DNA extractions were performed by using Dneasy Tissue Kit (Qiagen) according to the manufacturer’s recommendation. 2.3. Pulsed field gel electrophoresis DNA fingerprint by PFGE was performed as previously described [8,12,13]. 2.4. Genotyping of prn and pPtxA1 genes Genotyping of the genes encoding PRN (regions I and II) and the PT subunit S1 was performed as previously described [13,14].

2.5. Western blot analysis Western blot analysis was performed as described by Weber et al. [8]. 2.6. Serotyping Fim2 and Fim3 detection was performed by using monoclonal antibodies as described previously by Guiso et al. [15]. 2.7. Adenylate cyclase activity Adenylate cyclase activity was measured as previously described [16]. One unit corresponds to 1 nmol of cAMP formed per minute at 30 ◦ C and pH 8. 2.8. Pyrosequencing Pyrosequencing of the 2 genomes and analysis of the sequences were carried out by using a 454GS-FLX NextGen sequencing platform (Roche Diagnostics GmbH, Cogenics). The 2 samples were simultaneously sequenced in one GSFLX run using one 70 mm × 75 mm Pico-Titer plate device (Roche Diagnostics GmbH) and one GS LR-70 sequencing kit (Roche Diagnostics GmbH), as previously described [9]. 2.9. DNA microarray hybridization The DNA microarray, representing 91% of the predicted sequence of B. pertussis reference strain Tohama I, is similar to the one previously described [7,9]. Purified gDNA of isolates was labelled with either Cy3- or Cy5-dCTP, pooled with labelled DNA of the reference Tohama I strain, and hybridized on pre-treated slides. For each isolate, a dye-swap experiment was realised simultaneously. After hybridization, slides were washed with the appropriate buffers and then scanned with the GenePix4000B device. 2.10. Cell growth conditions 2.10.1. J774-A1 macrophage cells J774-A1 cells were cultured in RPMI1640 + Glutamax (GibcoBRL) supplemented with 10% FCS (DAP), 100 U/ml penicillin, 100 ␮g/ml streptomycin and 250 ng/ml amphotericine (GibcoBRL), 10 mM HEPES (Gibco-BRL) and 1 mM Na pyruvate (Gibco-BRL) in 75 ml tissue culture flasks (Corning). Cells were maintained in a 5% CO2 atmosphere at 37 ◦ C, as previously described [17]. 2.10.2. HTE epithelial cells Cells were plated in tissue culture trays coated with collagen (Polylabo) and cultured in DMEM-Glutamax (Gibco-BRL) supplemented with 2% UltroserG, 100 U/ml penicillin, 100 ␮g/ml streptomycin and 250 ng/ml amphotericine (Gibco-BRL), in a 5% CO2 atmosphere at 37 ◦ C, as previously described [17].

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2.10.3. Cytotoxicity assays Bacterial cytotoxicity towards J774-A1 cells was measured as previously described [17]. Briefly, bacteria were added to cells at 100:1 bacteria-to-cell ratio. Infected cells were incubated at 37 ◦ C, in the presence of 5% CO2 for 8 h in 96-well tissue culture trays. Cytotoxicity was determined by Cytotox 96 assay (Promega) which measure lactate deshydrogenase activity released in the medium every 2 h. 2.10.4. Invasion assays Invasion assays were done for HTE epithelial cells as previously described [17]. Briefly, bacteria were added to cells at a 100:1 bacteria-to-cell ratio. Infected cells were incubated at 37 ◦ C, in the presence of 5% CO2 for 7 h in 24-well tissue culture trays coated with collagen. After 5 h of incubation, cells were washed extensively and incubated for 2 additional hours with Gentamycin (Sigma). 2.10.5. Murine intranasal model of infection All procedures involving animals were conducted in agreement with the Institut Pasteur animal care and use committee guidelines. Bacteria were grown on BGA for 72 h and again for 24 h before inoculation. Four-week-old female Swiss mice (CERJ, Le Genest-St-Isle, France) were used to determine the behavior of the bacteria in the respiratory tract of young mice, as described by Khelef et al. [18]. Briefly, mice were infected intranasally with 50 ␮l of bacterial suspension of the reference strain Tohama I or of each isolate. Infected mice were sacrificed by cervical dislocation 2 h after infection and at different time points (Day 5, Day 8, Day 12 and Day 22; 4 mice per time point). The lungs were rapidly removed, homogenized in saline solution and serial 10-fold dilutions of the homogenates were used to determine the CFU on BGA plates. For active immunization, Balb/C mice were used as described previously [19,20]. Briefly, all vaccines were provided by GlaxoSmithKline Biologicals (Rixensart, Belgium). Four-week-old female Balb/C mice (CERJ, Le Genest-St-Isle, France; n = 5) received two subcutaneous doses (1/4 normal human dose) of the respective vaccine at 2 weeks intervals. Control group was given control solution. Two weeks after the second immunization, 50 ␮l of bacterial suspension containing 2–5 × 106 Colony Forming Unit (CFU) of bacterial suspension was instilled intranasally under light ether anesthesia. Mice were killed by cervical dislocation 2 h later or 5 and 8 days after infection. The lungs were rapidly removed, homogenized in saline, and serial 10-fold dilutions of the homogenates were used to determine the CFU on BGA plates. 3. Results 3.1. Description of the isolates The National French surveillance is a hospital-based surveillance and for this reason the majority of the isolates are collected on infants hospitalized for pertussis (http://www.invs.sante.fr/ surveillance/coqueluche/index.htm). They are mostly less than 3 months of age and not vaccinated. In 2007, we confirmed the identification of 71 B. pertussis isolates (http://www.pasteur.fr/sante/ clre/cadrecnr/bordet-index.html). They were collected all around France. The majority of the patients was less than 6 months of age and no or incompletely vaccinated. The analyses performed included classical bacteriological techniques and bacterial typing using Pulsed Field Gel Electrophoresis (PFGE). None of these isolates could be distinguished from previous circulating isolates. All isolates were included in PFGE group IV [12] and expressed the fimbrial protein type 3. All isolates were hemolytic and displayed adenylate cyclase activity, confirming the expression of adenylate cyclase-hemolysin toxin (AC-Hly), one of the major pertussis tox-

ins. The expression of other major toxins and adhesins, such as PT, FHA, and PRN, was examined by Western blotting. Among the 71 isolates analyzed, for the first time, a difference has been found in 5 (7%): one isolate (FR3749) was not expressing PT and four isolates (FR3693, FR3705, FR3708 and FR3793) were not expressing PRN. The characteristics of the isolates are shown on Table 1. The isolate FR3749 (PT− ) harbors a deletion of the ptx operon, including the structural genes encoding the five subunits of PT, as verified by PCR (data not shown). The sequencing of the PCR product targeting the repeated regions I and II of the prn structural gene of three PRN-deficient isolates revealed a type 2 prn gene. However, the sequence of the PCR product targeting region I and II indicated that an IS481 was inserted inside region II (Accession No. FJ480201). For the fifth isolate (FR3793), the sequence of the PCR product targeting region I revealed a deletion of the BP1053 gene coding for an IS1663, located upstream the prn gene BP1054, and of the first part of the prn gene (Accession No. FJ480200). The adenylate cyclase activity (50 U/ml), as measured in a bacterial suspension of FR3749 (PT− ) grown in Bordet Gengou medium, was similar to that of the reference strain Tohama I (60 U/ml) but reproducibly higher than that (14 U/ml) of FR3693 (PRN− ). This observation correlates with data we obtained previously with a PRN− mutant [18] and suggests that these isolates are probably less virulent than PRN-expressing isolates. In order to investigate whether other characteristics can be associated with these PT- or PRN-deficient isolates, we sequenced the genome of two of them, FR3749 (PT− ) and FR3693 (PRN− ) by using 454GS-FLX pyrosequencing. 3.2. Comparative genomics of PT- or PRN-deficient isolates with the reference strain Tohama I One sequencing run, using the 454 Roche Technology, was performed with the two isolates FR3749 (PT− ) and FR3693 (PRN− ) simultaneously. Results are summarized in Table 2. Sequences of about 240 nucleotides were obtained for the two isolates. Considering a genome size of 4 Mb for B. pertussis, the results we obtained gave a 14× and 11× coverage for FR3749 (PT− ) and FR3693 (PRN− ), respectively. The 1407 and 1630 contigs obtained for FR3749 and FR3693, represent 90% of each genome. The contigs were mapped with the sequenced genome of the reference strain Tohama I, the only annotated genome available for B. pertussis [21]. We noticed 2371 and 2045 sequences that were defined as unmapped on the genomes of FR3749 (PT− ) and FR3693 (PRN− ), respectively. These sequences represent approximately 45 kb, corresponding to 4 Regions of Difference (RD) previously identified in the genome of several recent B. pertussis clinical isolates [9], and also in the sequenced genomes of B. parapertussis and B. bronchiseptica, but not in the genome of the reference strain Tohama I [9,22,23]. Three other main RD, BP0911-BP0934, BP1135-BP1141 and BP1948-BP1966, were found to be deleted in the genome of the Table 2 454GS-FLX pyrosequencing data.

Mean sequences size Number of reads Nb bases Sequences assembled Sequences as repeats Unmapped sequences Number of contigs Average size of contigs Assembled bases Percentage of the Tohama I reference sequence Coverage

FR3749 (PT− )

FR3693 (PRN− )

240 215,237 51,163,550 196,442 16,424 (7.6%) 2371 1407 3167 3,608,866 90%

240 182,069 43,265,864 165,759 14,265 (7.8%) 2045 1630 2851 3,587,902 90%

14×

11×

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Table 3 Characteristic of region of difference from BP3780 to BP3811. Gene.ID BP3780 BP3782 BP3783 BP3784 BP3785 BP3786 BP3787 BP3788 BP3789 BP3790 BP3791 BP3792 BP3793 BP3794 BP3795 BP3796 BP3797 BP3798 BP3799 BP3800 BP3801 BP3802 BP3803 BP3804 BP3805 BP3806 BP3807 BP3808 BP3809 BP3810 BP3811

Gene

ptxA ptxB ptxD ptxE ptxC ptlA ptlB ptlC ptlD ptlI ptlE ptlF ptlG ptlH

argJ

Position (length)

Function

3,986,307–3,986,792 (486 bp) 3,987,011–3,987,991 (981 bp) 3,988,258–3,989,067 (810 bp) 3,989,107–3,989,787 (681 bp) 3,989,781–3990239 (459 bp) 3,990,212–3,990,613 (402 bp) 3,990,696–3,991,379 (684 bp) 3,991,435–3,991,743 (309 bp) 3,991,762–3,992,076 (315 bp) 3,992,073–3,994,547 (2475 bp) 3,994,555–3,995,946 (1392 bp) 3,995,943–3,996,128 (186 bp) 3,996,107–3,996,808 (702 bp) 3,996,805–3,997,626 (822 bp) 3,997,607–3,998,731 (1125 bp) 3,998,724–3,999,743 (1020 bp) Complement (3,999,949–4,000,839) (891 bp) 4,000,982–4001875 (894 bp) 4,001,958–4,002,332 (375 bp) 4,002,310–4,003,719 (1410 bp) 4,003,712–4,005,094 (1383 bp) 4,005,429–4,007,033 (1605 bp) 4,007,111–4,008,082 (972 bp) 4,008,093–4,009,019 (927 bp) 4,009,027–4,009,413 (387 bp)

Transposase (15 others) Putative exported protein (216 others) Pertussis toxin subunit 1 precursor Pertussis toxin subunit 2 precursor Pertussis toxin subunit 4 precursor Pertussis toxin subunit 5 precursor Pertussis toxin subunit 3 precursor Pertussis toxin transport protein Pertussis toxin transport protein (1 other) Putative bacterial secretion system protein (5 others) Putative membrane protein (160 others) Putative bacterial secretion system protein (5 others) Putative bacterial secretion system protein (5 others) Putative bacterial secretion system protein (5 others) Putative bacterial secretion system protein (5 others) Putative bacterial secretion system protein (5 others) Putative membrane protein (160 others) AraC family regulatory protein Conserved hypothetical protein (440 others) Putative membrane protein (160 others) Conserved hypothetical protein (440 others) Putative extracellular solute-binding protein (6 others) Putative transport system permease protein (4 others) Putative transport system permease protein (4 others) N-terminal region of a probable ABC transporter, ATP-binding protein (Pseudogene) Transposase (215 others) Arginine biosynthesis bifunctional protein Conserved hypothetical protein (440 others) Conserved hypothetical protein (440 others) Transposase (215 others) Transposase (215 others)

Complement (4,009,410–4,010,360) (951 bp) 4,010,530–4,011,756 (1227 bp) 4,011,956–4,012,822 (867 bp) 4,012,815–4,013,777 (963 bp) 4,013,878–4,014,828 (951 bp) 4,014,927–4,015,877 (951 bp)

two isolates presently sequenced, as previously reported for recent clinical isolates circulating in areas of the world with high vaccination coverage in children [9]. FR3749 (PT− ) harbors an additional deleted RD (BP3780-BP3811), the characteristics of which are presented in Table 3. This RD, delimited by transposases, contains the entire ptx operon, i.e. genes encoding PT subunits S1 to S5, genes involved in PT secretion (ptlA to ptlH) and other few genes encoding proteins involved in transport, binding or arginine biosynthesis. These data confirmed our previous results generated by PCR. King et al. [23] recently observed that some isolates associated with the Dutch epidemics possess a deletion of the BP3314-BP3322 genes. The two isolates FR3749 (PT− ) and FR3693 (PRN− ) do not present such a deletion. The genomes of the three isolates FR3749 (PT− ), FR3693 (PRN− ) and FR3793 (PRN− ) were analyzed by microarray for comparative genomic hybridizations versus the reference strain Tohama I (ArrayExpress Accession: E-MEXP-1871). We confirmed all data obtained after 454 pyrosequencing of FR3749 (PT− ) and FR3693 (PRN− ) genomes. Similar result was obtained for FR3793 (PRN− ). Moreover, an additional deletion was found for this isolate corresponding to RD from BP3050 to BP3054, delimited by transposase and containing 1 pseudogene, 2 putative membrane proteins, 1 putative oxydoreductase and 1 putative transpeptidase, and the deletion of BP1054 gene encoding pertactin. Interestingly, Newbler software designed 16424 (7.81%) and 14265 (7.82 %) reads as repeats in the genome of FR3749 (PT− ) and FR3693 (PRN− ), respectively. We observed that most of these repeats possess a positive blast to insertion sequences (IS). When blast hits of more than 200 nucleotides are considered, most of the repeated sequences are identified as IS, which accounts for 67.4% in FR3749 (PT− ) and 68.3% in FR3693 (PRN− ). As expected, the majority of the IS gave a positive blast with the sequence of IS481: 89.8% in FR3749 (PT− ) and 89.3% in FR3693 (PRN− ). Furthermore, IS1663

and IS1002 represent 7.5% and 2.2% of the repeated sequences in the genome of FR3749 (PT− ) and 8.5% and 2.6% in the genome of FR3693 (PRN), respectively. Considering the coverage obtained for each strain [i.e.14× for FR3749 (PT− ) and 11× for FR3693 (PRN− )] the estimated copy number of each IS was greater than reported on the genome of the reference strain Tohama I by Parkhill et al. [21]. No IS1001 was detected in the genome of the two isolates. Thus, 32.6 % and 31.7 % of the repeated sequences did not correspond to any known IS in the genome of FR3749 (PT− ) and FR3693 (PRN− ), respectively. They could correspond to repeated sequences located inside structural genes. When comparing the sequences of the two isolates with that of the reference, we observed some sequence differences (Supplementary File). We only considered the differences that were observed in all the reads, corresponding to a same region. Moreover, we also focused on the polymorphism of genes involved in the pathogenicity of B. pertussis (Table 4). We found 263 differences between the genome sequence of the reference strain and that of FR3749 (PT− ) and 259 for FR3693 (PRN− ), with 208 being common to both isolates (Supplementary Table). Among the 208 shared differences, 71 were found out of coding sequences (CDS), among which 70% accounted for differences located inside IS481 sequence (principally in the regions located between the inverted repeat and the transposase encoding sequence). Among the specific differences observed in the genome of FR3693 (PRN− ), we noticed differences located in the genes encoding subunits S1, S2 and S3 of PT. One of the mutations found out of CDS corresponds to a mutation in PT promoter polymorphism in S1 and S3 has been well studied [10]. As expected, the isolate FR3693 expresses a ptxA1 and ptxC2 type. The allele ptxS3-B harbors a silent point difference which was previously described in the genome of isolates circulating in The Netherlands, United Kingdom and United States, collected from 1980 to 1999 [24,25]. The non-silent differ-

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Table 4 Characteristics of the genes encoding the PT and PRN of FR 3749 (PT− ) and FR3693 (PRN− ). FR 3749

FR 3693

Tohama I

Reference

PT

Promoter ptxP Subunit S1 ptxA Subunit S2 ptxB Subunit S4 ptxD Subunit S5 ptxE Subunit S3 ptxC PT detection

No gene No gene No gene No gene No gene No gene Absence

ptxP3 ptxA1 A (45) ptxD ptxE ptxC2 Presence

ptxP1 ptxA2 G (45) ptxD ptxE ptxC1 Presence

[23] [25] [26] This study This study [25] Western blot

PRN

Region I Region II PRN detection

Type 2 Type 2 Presence

Type 2 Type 2 (insertion of an IS481) Absence

Type 1 Type 1 Presence

[34] [34] Western blot

ence that results in an amino-acid substitution (S vs G at position 45 of the amino acid sequence) in S2 subunit was also present in the genomes of the three recent epidemiological isolates that we previously sequenced [9] and was also recently described in an isolate circulating in Australia [26]. The promoter of the ptx operon is a prptx-P3 type, similar to the one harbored by the isolates recently collected in The Netherlands and in France [23]. 3.3. Cytotoxic properties of the PT-deficient and PRN-deficient isolates towards murine macrophages FR3749 (PT− ) and FR3693 (PRN− ), as well as the three other PRN− isolates (FR3705, FR3708, FR3793) were cytotoxic for murine macrophages J774-A1. No significant difference was observed, as compared to the reference strain Tohama I (data not shown). Even PRN-deficient isolates with less AC activity were cytotoxic. These isolates conserve some of their virulent characters. 3.4. Invasive properties of PT-deficient and PRN-deficient B. pertussis isolates on human tracheal epithelial cells We investigated the invasive properties of FR3749 (PT− ) and FR3693 (PRN− ), as compared to that of the reference B. pertussis Tohama I strain, in HTE cells. As shown in Fig. 1, FR3749 isolate displayed invasive properties comparable with that of the reference strain Tohama I. However, FR3693 (PRN− ) and FR3793 (PRN− ) were significantly more invasive, which is in line with previous data obtained with PRN− and AC-Hly-mutants that are more invasive in epithelial cells [17].

Fig. 1. Invasion of HTE cells by FR3693 (PRN− ), FR3793 (PRN− ), FR3749 (PT− ). Each strain was added to an individual well of a 24-well tissue culture plate containing approximately at least 105 cells/ml in order to reach a bacterium to cell ratio of 100:1. The invasion of HTE cells was assayed as described in Section 2. Invasion potential of each isolate was compared to that of the B. pertussis reference strain Tohama I. Results represent the means and standard deviation of at least three experiments. The symbol (*) indicates p < 0.05 versus the B. pertussis reference strain Tohama I.

3.5. PT-deficient and PRN-deficient B. pertussis isolates in the murine respiratory infection model FR3749 (PT− ) was unable to cause a lethal infection in 4-weekold young Swiss mice, even at a challenge dose of 109 CFU, whereas FR3693 (PRN− ) and FR3793 (PRN− ) had an LD50 similar to that of the reference strain Tohama I (2–5 × 108 , data not shown).

), FR3749 (PT− ( )), FR3693 (PRN− ( )); Fig. 2. Bacterial lung colonization observed in mice after intranasal challenge. (A) B. pertussis Tohama I ( ), FR3749 (PT− ( )), FR3793 (PRN− ( )). Groups of 4-week-old Swiss mice (n = 4) were challenged with 5 × 106 CFU of B. (B) B. pertussis Tohama ( pertussis isolates. Bacterial lung colonizations were measured at the indicated time points after challenge, and the results are expressed as the weighted mean CFU per lung (log10 -transformed). The plots show the geometric means for 4 mice per time point.

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Fig. 3. Bacterial lung colonization in mice after intranasal challenge with (A) B. pertussis FR3749 (PT− ) or (B) B. pertussis FR3693 (PRN− ); (C) B. pertussis reference strain. Intranasal challenge with 5 × 106 CFU of Bordetella pertussis isolates was performed in 7-week-old Balb/C control mice ( ) or in mice vaccinated with Diphtheria). Mice vaccinated with Diphtheria-Tetanus-Haemophilus b-three component Pertussis acellular vaccine Tetanus-Haemophilus b-Pertussis whole cell (Pw) vaccine ( ). Mice vaccinated with Diphtheria-Tetanus-two component Pertussis acellular vaccine (Pa) ( ). Mice vaccinated with Diphtheria-Tetanus-three (Pa) ( ). Bacterial lung colonization was measured at the indicated time points after challenge, and the results are expressed as component pertussis acellular vaccine (Pa) ( the weighted mean CFU per lung (log10 -transformed). The plots show the geometric means for 5 mice per time point. Numbers refer to the number of infected animals/number of animals sacrificed at each time point.

As shown in Fig. 2, colonization of the lungs by FR3749 (PT− ) was significantly different from that of the reference strain Tohama I. FR3749 (PT− ) was not able to colonize efficiently the respiratory tract of the animals after infection and did not multiply. On the contrary, the two PRN-deficient isolates revealed the same infection profile as the Tohama I strain, being able to multiply and to colonize the respiratory tract of young mice (Fig. 2). 3.6. Comparison of pertussis vaccine-induced immunity against infection due to PT-deficient and PRN-deficient B. pertussis isolates in the murine respiratory infection model The murine respiratory model is a convenient and reproducible tool to analyze the effectiveness of pertussis vaccine-induced immunity in promoting lung clearance of B. pertussis isolates [20,27]. However, the first step is to ensure that the different isolates reveal a similar infection profile in mice. The colonization of the two clinical isolates FR3749 (PT− ) and FR3693 (PRN− ) after intranasal challenge of adult mice is illustrated in Fig. 3. FR3749 (PT− ) was unable to colonize and multiply in the respiratory tract of naïve adult animals and was eliminated from the respiratory tract (control). The reference strain Tohama I and PT expressing clinical isolates were previously shown to behave differently [19,27] i.e. they were able to multiply in the respiratory tract of naïve adult mice. FR3693 (PRN− ), although behaving like reference strain Tohama I after infection of 4-week-old mice, was found to colonize the respiratory tract of the naïve adult animals (control) but was not able to multiply. Therefore, this isolate differs in that respect from PRN expressing isolates [19,27]. As further illustrated in Fig. 3, the immunity induced by either the Pw combined vaccine TritanrixTM or the trivalent combined vaccine InfanrixTM or bi or trivalent in house-vaccines combined to diphtheria and tetanus vaccines was very effective in promoting lung clearance of both isolates. No difference was noticed between the different vaccines. However, the bivalent vaccine has been shown to induce only partial protection against reference strains expressing both PT and PRN [20,27]. 4. Discussion In France, Pa vaccines are used for boosters since 1998, for most of the primary vaccinations since 2002, and for all vaccinations

since 2005. There are no more commercial Pw vaccines in France. Adolescent booster is recommended since 1998, and adult booster since 2004. In recent studies [7,9], we showed that herd immunity induced by Pw vaccination of young children during 40 years has led to the control of isolates that are similar to the vaccine strains. This phenomenon has not been observed in the Senegal, a region with low vaccine coverage [11]. Pa vaccines are now replacing Pw vaccines in France. In contrast with Pw vaccines that are targeting the bacterium as a whole, Pa vaccines are targeting the virulence of B. pertussis. This virulence does not seem to have changed over time, both in terms of toxin production and pathogenicity, as evaluated in animal and cellular models [28]. Since vaccine coverage is increasing in France thanks to the addition of Pa boosters for adolescents and adults, one may expect increased circulation of isolates expressing less or no vaccine antigens, or exhibiting higher expression of non-vaccine antigens implicated in virulence. In the present study, in support of our previous hypothesis [9], we report for the first time the emergence of B. pertussis isolates deficient in the two vaccine antigens PT and PRN. All PT-deficient and PRN-deficient isolates of this study were collected in France in 2007 in newborns younger than 6 months. PT is an ADP-ribosylating toxin that elicits biological effects through covalent modification of host proteins. This antigen induces protective immunity and is, therefore, included in all commercially available Pa vaccines. PT plays an important role in the development of disease but is not sufficient to cause pertussis. Indeed, although PT is not produced by B. parapertussis, this pathogen is able to induce pertussis-like illness comparable to that associated with infection by B. pertussis. The PT-deficient isolate (characterized by deletion of the entire ptx operon) analyzed in this study induced pertussis symptoms like in a B. parapertussis infection. Indeed, it did not cause hyperlymphocytosis in the infected infant, consistent with the absence of PT expression. We observed that this isolate is still able to induce macrophage apoptosis and to invade human epithelial cells, similar to PT-expressing isolates [17]. Nevertheless, we confirmed that this isolate was not lethal in the intranasal murine model and was not able to multiply in the respiratory tract of young or adult mice. PRN is a surface protein, which belongs to a class of autotransporter proteins that undergoes autoproteolytic processing. PRN contains a RGD motif (Arg-Gly-Asp) involved in the attachment of B. pertussis to mammalian cells. An IS481 inserted in the prn

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gene is responsible for the non-expression of PRN in three isolates (FR3693, FR3705 and FR3708) and the partial deletion of the prn gene in another isolate (FR3793). The PRN-deficient isolates caused infection in infants that was not distinguishable from a pertussis infection induced by PRN-expressing wild-type isolates, although exact clinical comparison was not possible a posteriori. However, differences were evidenced when using cellular or animal models. It was shown that PRN-deficient isolates, as PRN-constructed mutants [17], express less adenylate cyclase activity in bacterial suspensions, which again suggests an interaction between AC-Hly and PRN; PRN-deficient isolates are more invasive in epithelial cells, which is indicative of a longer persistence. Of note, PRN-deficient isolates are able to multiply in the respiratory tract of young mice but not in the respiratory tract of adult mice, suggesting a decrease in virulence in adults. Further investigations will be necessary to address these hypotheses. Using the murine intranasal challenge model, it was shown that immunization of mice prior to infection with PT- or PRN-deficient isolates with Pw and Pa-2 or Pa-3 vaccines lead to an early clearance of bacteria from the respiratory tract of mice as compared to the non-vaccinated animals. No significant differences are observed between the different vaccines tested in the present study. This result is different for the one obtained when the infection is performed with the reference strains 18,323 or Tohama I or isolates actually circulating and expressing PRN or PT. In fact, whatever the clinical isolates used to infect mice, we previously showed that French Pw vaccine and Pa-3 vaccine-induced immunity clears the bacteria more efficiently than Pa-2 vaccine immunity [19,27], Guiso et al. (manuscript in preparation). The circulation of such isolates might be the source of biological diagnosis problems, particularly in serological analysis rather than real time PCR. Indeed, serological diagnosis is based on the detection of anti-PT antibodies, which means that infections caused by PT-deficient isolates in adults might escape diagnosis. The genome of two of these isolates was almost entirely sequenced (90%) and, besides the genes involved in PT or PRN expression, they present the same genetic characteristic as other isolates circulating today regarding polymorphism of virulencerelated genes. They are also characterized by the same genetic content (RD). However, one striking observation is the high number of IS in their genome. Since the sequencing of a representative of the three species of B. pertussis, B. parapertussis and B. bronchiseptica by Parkhill et al. [21], all genome comparison studies have pointed out the important role of IS elements in the evolution of Bordetella species. Most rearranged or deleted regions in recent comparative genomic studies [7,9,23,29–31] are flanked by one or two IS. IS element proliferation is a key feature in genome reduction [32,33]. Species that have recently evolved as specialized pathogens often harbor higher number of IS than ancestor species, as observed for B. pertussis and B. parapertussis, in contrast to B. bronchiseptica. The proliferation of IS is expected to generate high mutation rate, large deletions, gene inactivation and massive reduction of the genome. The important number of IS copies observed in the presently described isolates may be the origin of genomic changes, leading to the inactivation of additional genes and explaining differences with previously sequenced genomes. Furthermore, isolates harboring high number of IS might be more instable and more difficult to collect. In conclusion, the present study describes the circulation of vaccine antigen-deficient B. pertussis isolates in a region with high Pa vaccine coverage in children and adolescents. Analysis of these isolates suggests that they are still virulent in infants but might be less virulent in adults, which might lead to clinical underreporting of pertussis cases. Furthermore, serological diagnosis, actually based on anti-PT antibodies (the only one specific to B. pertussis infection), will also suffer from under-

reporting if PT-deficient isolates are circulating. All isolates presently described were collected from non-vaccinated infants or infants that received only one dose of vaccine, indicating that these isolates are still virulent for immuno-compromised subjects. As many infants are infected by adults (http://www. invs.sante.fr/surveillance/coqueluche/index.htm) [5], increase in adult vaccine coverage is expected to result in a decrease in contaminated infants. Several questions may arise from our observation: Will the circulation of such isolates increase in correlation with the increase of vaccine coverage? Preliminary analysis of the isolates collected in 2008 and beginning of 2009 indicate that there are again some isolates not expressing PRN circulating. Since PRN is an outer membrane protein, contrary to PT and FHA that are secreted proteins, it could be more sensitive to herd immunity. Will we still be able to collect such isolates? Will they compensate their deficit in PT or PRN by over-expressing other virulence factors or expressing new ones? The diagnosis tools will probably need to be adapted to such evolution. Our results emphasize the usefulness of a surveillance system and the relevance of the collection of clinical isolates on the way to address these issues. Acknowledgements We are grateful to Gyslaine Guigon for her help in bioinformatics analyses and Sophie Guillot, and Marie-Laure Rosso for discussions. We thank the members of the Hospital-based surveillance, RENACOQ: Isabelle Bonmarin and Daniel Levy-Bruhl from the Institut de Veille Sanitaire and all the clinicians and microbiologists of the 43 peaditric hospitals involved in the surveillance: Dr Theveniau, Dr Chardon (Aix-En-Provence); Pr Garnier, Dr La Scola (Marseille); Pr Guillois; Dr Leclercq (Caen); Dr Guillot, Dr Paris (Lisieux); Dr Romanet, Dr Biessy (La Rochelle); Pr Huet, Dr Duez (Dijon), Dr Dagorne, Dr Vaucel (Saint Brieuc); Dr Hoen, Dr Couetdic (Besanc¸on); Dr de Parscaud, Pr Picard (Brest); Dr Sarlangue, Dr Lehours (Bordeaux); Dr Reygrobellet, Dr Jean Pierre (Montpellier); Dr Bonnemaison, Dr Lanotte (Tours); Dr Bost-Bru, Dr Croize, Dr Pelloux (Grenoble), Pr Mouzard, Dr Gibaud (Nantes); Dr Bentata-Durupt, Dr Barthez-Carpentier (Orléans); Dr Leneveu, Dr Le Coustumier, Dr Wilhems, Dr Février (Cahors); Dr Savagner, Pr Cottin (Angers); Dr Chomienne, Dr Laurens (Cholet), Pr Morville, Dr Brasme (Reims); Dr Monin, Dr Weber (Nancy); Pr Martinot, Pr Courcol (Lille); Dr Blanckaert, Dr Verhaeghe (Dunkerque); Dr Parlier, Dr Darchis (Compiègne), Pr Labbe, Pr Bonnet (ClermontFerrand); Dr Fischback-sheftel, Dr Kiesler (Strasbourg); Dr De Briel, Dr Kretz (Colmar); Pr Floret, Pr Etienne, Dr Quaglia (Lyon); Dr Bonardi, Dr Marmonier (Le Mans), Pr Grimprel, Pr Garbargchenon, Dr Moissenet (Trousseau Hospital, Paris); Pr Bourrillon, Pr Bingen, Dr Bonacorsi (R. Debré Hospital, Paris); Pr Cheron, Dr Descamps, Dr Ferroni (Necker Hospital Paris); Pr Gendrel, Dr Raymond, Dr Poyard (Saint-Vincent-de-Paul Hospital, Paris); Dr Meunier, Dr Le Luan (Fécamp); Pr Mallet, Dr Lemeland, Dr Nouvellon, Dr Boyer (Rouen); Pr Eb, Dr Hamdad-Daoudi (Amiens); Dr Fortier, Dr Lefrand (Avignon); Dr Menetrey, Dr Denis, Dr Ploy (Limoges); Pr Gaudelus, Dr Poilane (Bondy); Dr Delacour, Dr Estrangin, Dr Aberrane (Créteil); Pr Carrière, Dr Prère, Dr Delmas (Toulouse); Dr Parez, Dr Valdes (Colombes); Dr Tara Maher, Dr Reveil (Charleville-Mezières). As well as Dr Grattard (St Etienne). This work was performed with the financial help of the Institut Pasteur Foundation, URA CNRS3012, and GlaxoSmithKline Biologicals, Rixensart, Belgium. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.vaccine.2009.07.074.

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