INFECTION AND IMMUNITY, Feb. 2009, p. 896–903 0019-9567/09/$08.00⫹0 doi:10.1128/IAI.00769-08 Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 77, No. 2
Immunodominance in Mouse and Human CD4⫹ T-Cell Responses Specific for the Bordetella pertussis Virulence Factor P.69 Pertactin䌤 Rachel M. Stenger,1* Martien C. M. Poelen,1 Ed E. Moret,2 Betsy Kuipers,1 Sven C. M. Bruijns,1 Peter Hoogerhout,1 Marcel Hijnen,3† Audrey J. King,3 Frits R. Mooi,3 Claire J. P. Boog,1 and Ce´cile A. C. M. van Els1 Laboratory of Vaccine Research, Netherlands Vaccine Institute,1 and Laboratory of Vaccine Preventable Diseases, National Institute for Public Health and the Environment,3 Bilthoven, and Medicinal Chemistry and Chemical Biology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht,2 The Netherlands Received 19 June 2008/Returned for modification 18 August 2008/Accepted 27 October 2008
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 CD4ⴙ T-cell epitopes on the P.69 Prn protein remain unknown. Here, a single I-Ad-restricted B. pertussis conserved CD4ⴙ 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 CD4ⴙ T cells in an HLA-DQ-restricted manner. During B. pertussis infection, the epitope was associated with a Th1-type CD4ⴙ T-cell response. Hence, this novel P.69 Prn epitope is involved in CD4ⴙ 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 CD4ⴙ T-cell mechanisms in preclinical and clinical vaccine studies. to pertussis resistance by regulating specific B-cell responses and by producing protective Th1- and Th17-type cytokines, such as gamma interferon (IFN-␥) (31, 39) and interleukin 17 (IL-17) (20), respectively. To further substantiate the role and maintenance of CD4⫹ T cells in protective pertussis immunity, however, more knowledge about specific targeted antigens and CD4⫹ T-cell epitopes is required. P.69 pertactin (P.69 Prn), the focus of this study, is regarded as an important antigen in pertussis vaccines. P.69 Prn is polymorphic, and among the 12 variants described to date (14), variation in P.69 Prn is essentially found only in two regions (region 1 and 2) composed of sequence repeats (34). Several studies have shown that P.69 Prn is important for immune protection against B. pertussis infection (7, 10, 45). Furthermore, acellular vaccines containing only pertussis toxin and filamentous hemagglutinin appear substantially less effective than vaccines containing P.69 Prn as well (16, 29, 37). In mice, passive and active vaccination showed that P.69 Prn confers protective immunity (24, 25). Taken together, these results strongly support a role for P.69 Prn in protection against whooping cough. To gain insight into the role of P.69 Prn as a CD4⫹ T-cell target, we established specific T-cell hybridomas (TCH) from primed BALB/c mouse lymph node cells. This approach led to the identification of an immunodominant B. pertussis conserved I-Ad-restricted CD4⫹ T-cell epitope in P.69 Prn that evokes strong proliferative and cytokine responses after infection or vaccination of BALB/c mice. Moreover, the P.69 Prn epitope is also associated with HLA-DQ-restricted CD4⫹ Tcell immunity in humans.
Whooping cough, or pertussis, is an acute infection of the upper respiratory tract that is most severe in young children (8). The disease is caused mainly by the gram-negative bacterium Bordetella pertussis. Although vaccination against pertussis has been very successful in reducing morbidity and mortality among children worldwide, during the past 2 decades the disease has reemerged in countries with well-implemented infant vaccination programs (2, 5, 9, 11, 15, 17). In addition to changes in transmission patterns and the emergence of new variants of B. pertussis (18, 35, 36, 48), waning immunity has been proposed as a major factor in the increasing incidence of whooping cough (19, 50). Knowledge of the mechanisms of immunity to pertussis is still incomplete (3, 30, 40). Antibody responses against virulence factors have been associated with protection in various clinical studies (7, 45), while, on the other hand, immunogenicity studies after pertussis vaccination failed to demonstrate an unequivocal correlation between titers of antibodies to vaccine antigens and vaccine efficacy (1, 13). Studies with mice have indicated that protective immunity to B. pertussis infection not only depends on humoral immunity but also requires a CD4⫹ T-cell response (26, 27, 30, 38). CD4⫹ T cells can add
* Corresponding author. Mailing address: Laboratory of Vaccine Research, Netherlands Vaccine Institute, Antonie van Leeuwenhoeklaan 11, 3721 MA Bilthoven, The Netherlands. Phone: 0031-30-2743999. Fax: 0031-30-2744429. E-mail:
[email protected]. † Present address: Macfarlane Burnet Institute for Medical Research and Public Health, Melbourne, Australia. 䌤 Published ahead of print on 17 November 2008. 896
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VOL. 77, 2009 TABLE 1. Synthetic 15-mer oligopeptides covering the N terminusa of B. pertussis P.69 Prn Peptide
a
TABLE 2. N-terminal sequencesa of the B. pertussis, B. parapertussis, and B. bronchiseptica P.69 Prn’sa
Amino acid sequence
B. pertussis P.69 Prn N terminus ...................1DWNNQSIVKTGERQHGIHIQGSDPGGVRTA P.69 Prn1–15 ......................1DWNNQSIVKTGERQH15 4 P.69 Prn4–18 ...................... NQSIVKTGERQHGIH18 7 P.69 Prn7–21 ...................... IVKTGERQHGIHIQG21 10 P.69 Prn10–24 .................... TGERQHGIHIQGSDP24 13 P.69 Prn13–27 .................... RQHGIHIQGSDPGGV27 16 P.69 Prn16–30 .................... GIHIQGSDPGGVRTA30 Amino acids 1 to 30.
MATERIALS AND METHODS Mice and cell suspensions. Female specific-pathogen-free BALB/c mice were purchased from Harlan and kept in-house under conventional conditions. All experiments were approved by the Animal Ethics Committee of the Netherlands Vaccine Institute (NVI). After section, single-cell suspensions of splenocytes and lymph node cells were produced by mechanical dissociation of organs through 70-m-pore-size nylon filters. Red blood cells in splenocyte suspensions were lysed with 10 mM KHCO3–0.1 mM EDTA for 2 min at 4°C. Splenocytes were resuspended in complete IMDM-10 (Iscove’s modified Dulbecco’s medium [Gibco-BRL] supplemented with 10% fetal bovine serum [HyClone] and penicillin, streptomycin, and L-glutamine [Pen/Strep/Glu; Gibco-BRL]). Lymph node cells were resuspended in complete IMDM-5 (Iscove’s modified Dulbecco’s medium supplemented with 5% normal mouse serum [Harlan] and Pen/Strep/ Glu). Isolation of PBMC. Peripheral blood was obtained after informed consent from HLA-oligotyped blood bank donors from a birth cohort associated with pertussis vaccination (S03.0015-x; Sanquin) and from two pertussis patients within 4 weeks after laboratory-confirmed diagnosis of B. pertussis infection (NVI-243). Peripheral blood mononuclear cells (PBMC) were isolated by centrifugation of buffy coat cells on Ficoll-Hypaque (Pharmacia Biotech, Uppsala, Sweden) and were used directly or after cryopreservation. Cell lines. The BW1100 cell line, a TCR␣⫺/⫺ variant of BW5147 (51), was used as a fusion partner for the production of TCH and was kindly provided by D. Canaday (Case Western Reserve University and University Hospitals, Cleveland, OH). The A20 BALB/c B-cell lymphoma cell line was obtained from the ATCC. The BW1100 and A20 cell lines were grown in complete DMEM-10 (Dulbecco’s minimal essential medium supplemented with 10% fetal bovine serum [HyClone] and Pen/Strep/Glu) (Gibco-BRL). B. pertussis strains and whole-cell vaccine (WCV). B1585 and B1675 are P.69 Prn2-containing isogenic B. pertussis strains derived from strain Tohama I (isolated in Japan in the 1950s) (23) and the Dutch vaccine strain 134 (isolated from an American patient in the 1940s), respectively. For the construction of these strains, nalidixic acid (Nal)-resistant derivatives of strain Tohama I and the Dutch vaccine strain 134, which both naturally harbor P.69 Prn1, were selected. The prn1 allele in these strains was exchanged with the prn2 allele by allelic exchange essentially as described by Stibitz et al. (44). Briefly, a PCR fragment containing prn2 was amplified from the Dutch clinical isolate B0345 with primers Prn-XbaI (5-GCTCTAGATGTAAAACGACGGCCAGTGGGCGGGCAGCG GGG-3) and Prn-EcoRI (5-GGAATTCCAGGAAACAGCTATGACCCCAGC TCCGGCGCCTCG-3) (the underlined sequence corresponds to bases 856 to 870 and 1080 to 1096 of the P.69 Prn sequence in GenBank accession number J04560, respectively). The PCR fragment, flanked by EcoRI and XbaI sites, was digested with the corresponding enzymes and cloned into the pKAS46 vector containing a kanamycin (Km) resistance gene (43). Escherichia coli SM10 was transformed with this plasmid construct and used as a donor in conjugation with the B. pertussis strains described above. Exconjugants were selected on BordetGengou-Nal-Km plates and checked for expression of the prn2 allele by DNA sequencing. For the preparation of WCV or the challenge inoculum, B1586, B1675, and B0613, a Dutch clinical isolate from 1995 expressing P.69 Prn2, were grown in Thijs medium (47). WCV was obtained after heat and formaldehyde inactivation of pelleted bacteria and addition of Al(OH)3 as an adjuvant. Recombinant P.69 Prn and synthetic peptides. Recombinant wild-type P.69 Prn1, Prn2, and Prn3 proteins from B. pertussis and deletion derivatives of P.69 Prn1 were expressed in Escherichia coli and were purified as previously described (22). Overlapping 18-mer peptides covering the entire B. pertussis P.69 Prn1
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Strain
P.69 Prn amino acid sequence
B. pertussis .....................DWNNQSIVKTGERQHGIHIQGSDPGGVRTASGT B. parapertussis..............DWNNQSIIKAGERQHGIHIKQSDGAGVRTATGT B. bronchiseptica ...........DWNNQSIIKAGERQHGIHIKQSDGAGVRTATGT a
Amino acids 1 to 33. The P.69 Prn7–24 peptide is shown in boldface.
protein, 15-mer peptides representing B. pertussis P.69 Prn1–15, P.69 Prn4–18, P.69 Prn7–21, P.69 Prn10–24, P.69 Prn13–27, and P.69 Prn16–30 (Table 1), and the Bordetella parapertussis- and Bordetella bronchiseptica-specific peptide P.69 Prn7–24 (Table 2) were prepared by solid-phase synthesis using N␣-(9-fluorenyl)methoxycarbonyl (FMOC)-protected amino acids and a Syro II simultaneous multiplepeptide synthesizer (MultiSyntech GmbH, Witten, Germany). The purity and identity of the synthesized peptides were assessed by reverse-phase high-performance liquid chromatography and mass spectometry. Immunization and intranasal infection of mice. For TCH production, four mice (ages, 3 to 4 weeks) were immunized subcutaneously at the groin with 0.016 international opacity units of B1675 WCV containing Al(OH)3 as an adjuvant. Vaccination was repeated on day 14, and on day 28 mice were challenged intranasally with 2 ⫻ 107 B1586 bacteria. On day 31, draining lymph nodes of the lungs were collected and pooled, and a lymph node cell suspension was prepared and used directly. For the generation of P.69 Prn1 immune splenocytes, five mice were immunized subcutaneously with 1.5 g recombinant P.69 Prn1 (rP.69 Prn1) containing AlPO4 as an adjuvant at day 0 and day 28. Section was performed on day 35. Spleens were dissected and used individually as splenocyte suspensions, either fresh or after freezing and storage at ⫺135°C. For the generation of B. pertussis immune splenocytes, four mice were infected intranasally with 2 ⫻ 107 CFU of B. pertussis strain B0613 per mouse. Ten days later, mice were sacrificed, and spleens were collected and tested individually. TCH generation. Lymph node cells from mice immunized with the B1675 WCV and challenged with B1586 bacteria were restimulated by culturing the cells at 5 ⫻ 106 cells/ml for 6 days in the presence of 0.5 g/ml rP.69 Prn2 and 20 ng/ml recombinant murine IL-7 (rmIL-7) (Tebu-bio) in IMDM-5. rmIL-2 (20 U/ml; BD Biosciences) was added on day 2. rmIL-7 and rmIL-2 were readded on day 4 in order to maintain their levels. On day 7, in vitro-restimulated lymph node cells and BW1100 cells were fused at a 1:1 ratio (⫾1 ⫻ 107 cells each) by using polyethylene glycol, as described by Canaday et al. (6). Cells were serially diluted in 96-well plates and incubated in DMEM-10 overnight. Then 2⫻ hypoxanthine-aminopterin-thymidine (Gibco-BRL) in DMEM-10 was added. If proliferation was observed, cells were transferred to 24-well plates, cultured in DMEM-10 with 1⫻ hypoxanthine-thymidine supplementation (Gibco-BRL), further expanded, and selected for CD3ε⫹ CD4⫹ CD8⫺ TCR␣⫹ TCR␥␦⫺ surface expression by flow cytometry. TCH assay. TCH were screened for antigen specificity by testing their ability to produce IL-2 upon stimulation. Briefly, 2 ⫻ 104 TCH cells were cocultured in triplicate or quadruplicate wells in DMEM-10 in the presence or absence of antigen (rP.69 Prn proteins or peptides) with 1 ⫻ 105 irradiated (20 Gy) A20 cells or naïve splenocytes, as antigen-presenting cells, in 200-l volumes in 96-well round-bottom plates (Greiner) at 37°C under 5% CO2. For major histocompatibility complex (MHC) restriction assays, 5 g of antibodies specific for class II molecules (I-Ad [clone 39-10-8], I-Ed,k,r,p [clone 14-4-4S], and I-Ad/I-Ed [clone 2G9]; all from BD Biosciences) was included in the incubation mixture. After 48 h, 100 l of the culture supernatant was assayed for IL-2 production by using an IL-2 sandwich enzyme-linked immunosorbent assay with a pair of murine monoclonal antibodies against IL-2 (JES6-5H4 and JES6-1A12; BD Biosciences). Results are expressed as mean IL-2 concentrations (in picograms per milliliter) ⫾ standard deviations (SD). Proliferation assay using immune splenocytes. Splenocytes from rP.69 Prn1immunized mice or intranasally infected mice were cultured at 1.5 ⫻ 105/150 l in 96-well round-bottom plates (Greiner) in the presence of P.69 Prn 18-mer oligopeptides in IMDM-10 or in medium only. On day 4, 100-l supernatant volumes were removed in order to determine cytokine concentrations. Then 0.5 Ci (18.5 kBq) [3H]thymidine (Amersham) was added to the wells, and cells were cultured for another 18 h. Cells were harvested, and [3H]thymidine incorporation was determined as counts per minute using a Wallac 1205 Betaplate liquid scintillation counter. Results are expressed as stimulation indices (SI) ⫾ SD from triplicate wells, calculated as (counts per minute of cultures in the
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presence of antigen)/(counts per minute of cultures in the presence of medium only). SI of ⬎1.5 are considered positive. Proliferation assay using PBMC. A total of 105 PBMC were incubated in complete AIM-V medium (AIM-V medium containing streptomycin, gentamicin, and L-glutamine) (Gibco-BRL) supplemented with 2% human AB serum (Harlan) at 150 l/well in 96-well round-bottom plates (Greiner) in the absence or presence of the relevant peptide(s) at 1 M or rP.69 Prn1 at 1 g/ml at 37°C under a 5% CO2 atmosphere. In blocking experiments, monoclonal antibodies specific for HLA-DR (1/10-diluted culture supernatant; B8.11.2) or for HLA-DQ (20 g/ml; SPV-L3) were included in the incubation mixture. At day 4, 100-l volumes were removed for the cytokine determinations. Then 0.5 Ci (18.5 kBq) [3H]thymidine (Amersham) was added to the culture 18 h before the cells were harvested. Counts per minute were determined, and results were calculated, as for the proliferation assay using immune splenocytes. Results are expressed as SI from triplicate wells for PBMC from infected individuals and as SI from octuple wells for PBMC from healthy individuals. SI of ⬎1.5 are considered positive. Cytokine profiling using Luminex technology. Concentrations of cytokines [IL-2, IL-4, IL-5, IL-10, IL-12(p70), IL-13, IL-17, granulocyte-macrophage colony-stimulating factor, tumor necrosis factor alpha (TNF-␣), and IFN-␥] in pooled murine or human culture supernatants obtained from in vitro-restimulated splenocyte or PBMC cultures, respectively, were determined using the Bio-Plex mouse or human Th1/Th2 and Th17 cytokine Luminex kits (Bio-Rad), respectively, according to the manufacturer’s instructions. Measurements and data analysis were performed with the Bio-Plex system in combination with Bio-Plex Manager software. Results are expressed in picograms per milliliter.
RESULTS Identification of an I-Ad-restricted CD4ⴙ T-cell epitope at the N terminus of P.69 Prn. To gain insight into the BALB/c P.69 Prn-specific CD4⫹ T-cell response, we set out to establish TCH by using primed BALB/c lymph node cells that were restimulated with rP.69 Prn2 in vitro. Several TCR␣⫹ CD4⫹ TCH were obtained, only one of which produced IL-2 after stimulation with rP.69 Prn2. Two other common pertactin variants, rP.69 Prn1 and rP.69 Prn3, were equally well recognized by this TCH, reflecting a specificity for a conserved part of P.69 Prn (Fig. 1A). When the TCH was tested against an rP.69 Prn deletion mutant lacking the first 19 amino acids of the adhesin (22), recognition was abrogated (data not shown). This indicated that the CD4⫹ T-cell epitope was located at the N terminus of the P.69 Prn molecule. Overlapping 15-mer oligopeptides covering the N terminus of P.69 Prn (Table 1) were then used to identify the fine specificity of the TCH response. A20 cells loaded with oligopeptides P.69 Prn7-21, P.69 Prn10-24, and P.69 Prn13-27, but not those loaded with P.69 Prn1-15, P.69 Prn4-18, or P.69 Prn16-30, were able to stimulate the TCH (Fig. 1B). Therefore, the core of the TCH epitope was fine mapped to amino acids 13 to 21 of P.69 Prn. To determine the MHC class II restriction of the response, rP.69 Prn1-loaded naïve splenocytes, used as antigen-presenting cells, were preincubated with anti-I-Ad, anti-I-Ed/I-Ad, or anti-I-Ed,k,r,p monoclonal antibodies prior to coculture with the TCH. As shown in Fig. 1C, only anti-I-Ad antibodies blocked the response, demonstrating that the N-terminal P.69 Prn epitope is naturally processed and presented in an I-Ad-restricted manner. Immunodominance of the N-terminal P.69 Prn epitope. Since the TCH approach yielded only one P.69 Prn fine specificity, immunodominance of the epitope was suggested. To substantiate our findings and to identify other possible specificities, Pepscan analyses were performed using immune splenocytes and smart peptide pools of overlapping 18-mer P.69 Prn peptides covering the mature P.69 Prn protein (data
FIG. 1. P.69 Prn cross-reactivity, epitope recognition, and MHC restriction of TCH cells. (A and B) A total of 2 ⫻ 104 TCH cells were cocultured with 1 ⫻ 105 A20 B cells as antigen-presenting cells in the presence of titrated amounts of rP.69 Prn1, -2, or -3 (A) or the indicated doses of overlapping 15-mer N-terminal P.69 Prn peptides (B). (C) Inhibition of IL-2 production by MHC class II-specific monoclonal antibodies in the TCH assay using rP.69 Prn1-loaded naïve BALB/c splenocytes as antigen-presenting cells. The concentrations of IL-2 in the supernatants were determined. Results are expressed as means ⫾ SD.
not shown). Splenocytes from mice vaccinated with P.69 Prn1 specifically proliferated at a high responder rate against distinct peptide pools, i.e., pools A, B, C, 2, 3, 4, 6, and 7 (data not shown), at 1 M/peptide. Subsequently, the proliferation of thawed rP.69 Prn1 immune splenocytes from the same mice in response to single peptides of these pools was measured. Three epitope regions, represented by peptides A2 and A3 (A2/A3;
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FIG. 3. Splenocytes recognizing the B. pertussis (Bp) P.69 Prn7–24 epitope are not stimulated by the B. parapertussis (Bpp) or B. bronchiseptica (Bb) homologue. BALB/c mice were vaccinated on day 0 and day 28 with B. pertussis-specific rP.69 Prn1 and were sacrificed on day 35. Proliferation was determined after restimulation of splenocytes with the indicated doses of P.69 Prn7–24 from B. pertussis or B. parapertussis/B. bronchiseptica. Results are expressed as mean SI ⫾ SD from four mice.
FIG. 2. Fine mapping of proliferative responses of immune splenocytes by using single peptides. BALB/c mice either were vaccinated (n ⫽ 5) on day 0 and day 28 with rP.69 Prn1 and sacrificed on day 35 (A) or were intranasally infected (n ⫽ 4) with 2 ⫻ 107 B. pertussis strain B0613 bacteria on day 0 and sacrificed on day 10 (B). A total of 1.5 ⫻ 105 splenocytes were incubated with 1 M single 18-mer P.69 Prn peptides, and [3H]thymidine incorporation was determined after 4 days of restimulation. Each bar represents the SI ⫾ SD for an individual mouse.
i.e., P.69 Prn7-30), C6 (i.e., P.69 Prn127-144), and B6/B7 (i.e., P.69 Prn79-102), in order of the relative magnitude of the response, were recognized by the majority of immunized mice (Fig. 2A). Upon repetitive testing, no single peptide from pool 4 that supported proliferation could be identified, suggesting that this pool does not contain any CD4⫹ T-cell epitope. The same epitope hierarchy (from greatest to least response, P.69 Prn7-30, P.69 Prn127-144, and P.69 Prn79-102) was found after vaccination with more-complex pertussis (whole-cell and acellular) vaccines, among two other minor peptide specificities, i.e., D6 (P.69 Prn175-192) and E4 (P.69 Prn211-228) (data not shown). In addition, we found that the response of splenocytes from mice 10 days after infection with B. pertussis strain B0613 was directed to the N-terminal epitope (A2/A3) only (Fig. 2B). Together, these results clearly show that the N-terminal P.69 Prn epitope is immunodominantly recognized by CD4⫹ T cells after vaccination as well as after infection.
Functional P.69 Prn sequence variation between Bordetella species. B. pertussis, B. parapertussis, and B. bronchiseptica are three closely related bordetellae associated with respiratory infections in humans and other mammals. Many of the common virulence factors, including P.69 Prn, are highly homologous between these three species. However, analysis of the respective P.69 Prn protein sequences revealed species-specific nucleotide polymorphisms in the immunodominant P.69 Prn7–24 epitope (Table 2). To investigate whether these mutations are immunologically relevant, we compared the stimulatory capacities of the B. pertussis-specific P.69 Prn7–24 peptide and its B. parapertussis and B. bronchiseptica counterparts by using splenocytes from B. pertussis P.69 Prn1-vaccinated mice. As shown in Fig. 3, the B. parapertussis and B. bronchiseptica P.69 Prn7–24 form was not recognized by the B. pertussis P.69 Prn1-immune splenocytes. This implies that the major CD4⫹ T-cell population involved in the B. pertussis P.69 Prn response cannot cross-protect during subsequent infection with B. parapertussis or B. bronchiseptica. CD4ⴙ T-cell epitope-specific cytokine production after B. pertussis infection. Since the N terminus of B. pertussis P.69 Prn evokes an immunodominant proliferative splenic CD4⫹ T-cell response, we further investigated whether this was associated with measurable cytokine production in ex vivo-restimulated splenocyte cultures. Such cytokine production would aid in the development of functional epitope-based assays to evaluate the type and maintenance of P.69 Prn-directed CD4⫹ T-cell populations in various B. pertussis immunization models. Accordingly, splenocytes from three BALB/c mice infected with B. pertussis strain B0613 were assayed for the induction of epitope-specific Th1-, Th2-, or Th17-type cytokine responses by a Luminex assay. As shown in Fig. 4, splenocytes from all infected mice produced high levels of IL-17 upon restimulation
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FIG. 4. P.69 Prn7–24-specific cytokine production of immune splenocytes. Three BALB/c mice were infected intranasally with 2 ⫻ 107 CFU of B. pertussis strain B0613/mouse on day 0 and were sacrificed on day 10. After restimulation of splenocytes for 4 days with the immunodominant P.69 Prn7–24 peptide at 1 M, cytokine production in the supernatant was determined using Luminex technology. The cytokine response of each mouse is represented by a different symbol.
with the dominant P.69 Prn7-24 epitope. In addition, elevated levels of IL-2 and IFN-␥, but only very limited production of IL-5, were observed. These results suggest that B. pertussis infection of BALB/c mice induces a combined Th1/Th17-type P.69 Prn7-24-specific CD4⫹ T-cell response. Detection of P.69 Prn N-terminus-specific CD4ⴙ T-cell responses in healthy HLA-DQ1ⴙ subjects. Extrapolation of experimental findings with mice to humans is difficult, and murine and human MHC processing and selection of CD4⫹ T-cell epitopes may differ widely. To establish the role of P.69 Prn7–24 in human anti-B. pertussis CD4⫹ T-cell immunity, we examined PBMC from 20 healthy HLA-typed individuals with a history of pertussis vaccination for the presence of a recall response against the P.69 Prn N terminus. A considerable proportion of individuals showed significant proliferation in response to the 15-mer P.69 Prn13–27, which was not associated with any particular HLA-DR allele (data not shown) but was associated with the presence of HLA-DQ1 (Fig. 5A). Hence, HLA-DQ1 may serve as a restriction element for the recognition of P.69 Prn13–27 by human CD4⫹ T cells. Since the preceding 15-mer oligopeptide, P.69 Prn10–24, also evoked PBMC proliferation from the same positive responders (data not shown), it is likely that both 15-mers P.69 Prn10–24 and P.69 Prn13–27 represent one shared human epitope sequence, which was in fact also the immunodominant BALB/c N-terminal P.69 Prn epitope represented by the 18-mers P.69 Prn7–24 (“A2”) and P.69 Prn13–30 (“A3”). To confirm this in-
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FIG. 5. The P.69 Prn N terminus is recognized by PBMC of healthy individuals in a HLA-DQ-restricted manner. (A) PBMC of HLAtyped donors (n ⫽ 20) were divided into two groups based on HLADQ1 expression. Proliferation of the PBMC in response to P.69 Prn13–27 was determined by [3H]thymidine incorporation after 5 days of stimulation and is expressed as the SI. Differences between the two groups were assessed by an unpaired two-tailed t test. (B) Proliferation of a cell line of a healthy HLA-DQ1-typed individual established against P.69 Prn13–27. Cells were expanded in vitro, allowed to rest, and tested against P.69 Prn1 (filled bar), P.69 Prn 18-mers (shaded bars), and P.69 Prn 15-mers (open bars) in the absence or presence of anti-HLA-DR (␣DR) or anti-HLA-DQ (␣DQ) monoclonal antibodies. Results are expressed as SI ⫾ SD.
terpretation, P.69 Prn13–27-stimulated PBMC from one healthy HLA-DQ1⫹ donor were allowed to rest and were subsequently restimulated with rP.69 Prn and various N-terminal P.69 Prn 15- and 18-mer oligopeptides. The proliferation pattern observed indicated that 18-mers P.69 Prn7–24 and P.69 Prn13–30 and 15-mers P.69 Prn10–24 and P.69 Prn13–27 represent the same shared human and murine CD4⫹ T-cell epitope (Fig. 5B). In addition, monoclonal antibodies specific for HLA-DQ, but not those specific for HLA-DR, completely inhibited the P.69 Prn13–27-directed response, confirming the involvement of the HLA-DQ1 allele as a restriction element (Fig. 5B). N-terminal epitope-specific proliferative and cytokine responses during acute B. pertussis infection. To assess the CD4⫹ T-cell response against the P.69 Prn N-terminal epitope during an acute symptomatic B. pertussis infection, PBMC from two patients suffering from whooping cough were restimulated with the 18-mer P.69 Prn13–30 and assayed for proliferative capacity and cytokine production. Patients’ PBMC proliferated in re-
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FIG. 6. Proliferation and cytokine production of P.69 Prn13–30stimulated PBMC from two B. pertussis-infected individuals. (A) Proliferation of PBMC of two pertussis patients in response to stimulation with P.69 Prn13–30 or P.69 Prn1. Results are expressed as SI ⫾ SD from individual patients. (B) Cytokine production in the supernatants of PBMC from the two pertussis patients was measured using Luminex technology 4 days after P.69 Prn13–30 restimulation. The cytokine production of each patient is given separately. The level of cytokine production by mock-stimulated PBMC was ⬍5 pg/ml for all cytokines measured except TNF-␣ (⬍45 pg/ml) and IL-2 (⬍10 pg/ml). Shaded bars, responses from patient 1; filled bars, responses from patient 2. GM-CSF, granulocyte-macrophage colony-stimulating factor.
sponse to P.69 Prn13–30 as well as to the whole P.69 rPrn1 protein (Fig. 6A). In addition, the P.69 Prn13–30-stimulated PBMC of the patients produced markedly elevated levels of TNF-␣ and small amounts of IL-2, IL-13, and granulocytemacrophage colony-stimulating factor relative to control levels (Fig. 6B). One patient’s PBMC also produced a significant amount of IFN-␥ in response to the epitope. No Th2 (IL-4 and IL-5)- or Th17 (IL-17)-type cytokines were detected in these stimulated cultures. The combined levels of IFN-␥ and IL-2 in the absence of detectable IL-4, IL-5, and IL-17 production are suggestive of a predominantly Th1 type cytokine profile. DISCUSSION Recognition of specific epitopes by CD4⫹ T helper cells is the basis for cellular defense mechanisms, contributing to the clearance of bacterial pathogens that have a facultatively intracellular lifestyle, such as B. pertussis. Indeed, CD4⫹ T cells have been shown to be protective in pertussis models of infection (26, 31). In this study, four major findings shed new light on the role of the pertussis adhesin P.69 Prn as a target for CD4⫹ T-cell immunity. First, we identified a novel BALB/c CD4⫹ T-cell epitope in B. pertussis P.69 Prn with the help of
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TCH technology. A CD4⫹ TCH recognized an N-terminal P.69 Prn epitope, represented by peptide P.69 Prn7–24, in an I-Ad-restricted manner. The most common B. pertussis pertactin variants—P.69 Prn1, P.69 Prn2, and P.69 Prn3—were equally efficiently recognized by the TCH cells, indicating that natural P.69 Prn polymorphism does not influence the efficiency of epitope processing and presentation. Second, this N-terminal P.69 Prn epitope was immunodominantly recognized by CD4⫹ T cells during an ongoing immune response in BALB/c mice, as measured by in vitro proliferation of, and cytokine production by, splenocytes. Subdominant P.69 Prn epitopes were P.69 Prn127–144, P.69 Prn79–102, P.69 Prn175–192, and P.69 Prn211–228, in order of the relative magnitude of their response. Third, our data indicated that B. pertussis-induced CD4⫹ T cells directed against the P.69 Prn N terminus will not likely cross-protect against infections with B. parapertussis or B. bronchiseptica. Fourth, the N-terminal P.69 Prn epitope recalled memory responses in a considerable proportion of healthy adults in an HLA-DQ1-restricted manner and was recognized during acute pertussis infection. Together, these findings designate the N-terminal P.69 Prn epitope as the first human and murine conserved pertussis-specific CD4⫹ T-cell epitope. We consider that a plausible explanation for the immunodominance of this epitope is its favorable MHC processing by antigen-presenting cells. Inspection of the X-ray structure of the first 539 amino acids of the P.69 Prn molecule (12) indicates that all five epitopes identified are located upstream of the first variable region and are partially masked by this region (Fig. 7A). However, in contrast to the subdominant epitopes, which are part of the relatively inaccessible, tightly folded -barrel structure, the immunodominant epitope is located at the extreme N terminus of the molecule and is flanked by two well-exposed -turns (G11ERQ14 and A30SGT33), which could be easy targets for lysosomal proteases. In fact, one cleavage event near sequence A30SGT33 would suffice to liberate the epitope region from the protein backbone, allowing it to become loaded onto nascent MHC class II molecules (Fig. 7B). Interestingly, the N-terminal P.69 Prn epitope was presented by both the murine and human MHC class II alleles I-Ad and HLA-DQ1, respectively. Such shared epitope specificity is in line with the functional homology between I-A and HLA-DQ molecules reported for other antigen models (46). In this study, the cross-species sharing of the N-terminal P.69 Prn epitope opened up the possibility of studying the quality of a CD4⫹ T-cell response with the same fine specificity in both experimental and clinical pertussis samples. For BALB/c mice 10 days after B. pertussis infection, a mixed P.69 Prn7–24-dependent cytokine response was found in splenocyte cultures, which produced IFN-␥ and IL-17, but little or no IL-4 and IL-5, after peptide stimulation. Although the phenotype of the splenocytes responsible for these cytokines needs to be assessed further, this peptide-dependent cytokine profile most likely matches the presence of P.69 Prn-specific Th1/Th17-type CD4⫹ T cells. On the other hand, for two pertussis patients, the CD4⫹ T-cell response to the epitope was associated not with an IL-17 response but with prominent in vitro production of TNF-␣ combined with IL-2 and, for one of the patients, with IFN-␥. TNF-␣ is an inflammatory cytokine that has been implicated in controlling B. pertussis through potentiating the
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FIG. 7. Schematic drawings of epitopes (colored) in the P.69 Prn structure (PDB code 1dab) (12). (A) Overview of the epitopes, showing that they are all N-terminal to region 1 (yellow). Colors represent epitopes as follows: red, A2/3; magenta, B6; green, C6/7; cyan, D6; blue, E2. (B) View of the N-terminal side and the immunodominant epitope A2/3, showing easy access for proteolytic enzymes. These drawings were prepared with Yasara (www.yasara.org).
bactericidal activity of macrophages and enhancing phagocytosis by neutrophils (20, 33). To be conclusive about an eventual P.69 Prn13–30-associated Th cytokine pattern in humans, these clinical observations need to be extended further, including more patients’ samples and cytokine analysis at the singlecell level. Nevertheless, our present data provide proof of principle that cytokine responses to the N-terminal P.69 Prn epitope can serve as a readout for B. pertussis-specific CD4⫹ T-cell programming in BALB/c mice and in humans. As such, the epitope could be of use in vaccine development. It has long been thought that Th1-type T-cell responses are most favorable in protective pertussis immunity (26, 32, 39) and that Th2-type T-cell responses should be avoided because of their unfavorable predisposition of infants to allergies (4, 28). More recently, the contribution of Th17-type T cells to protective vaccine responses against B. pertussis was reported (20). Hence, novel pertussis vaccines should be evaluated for their protective capacities as well as for the Th cytokine profiles they induce. In this context, the N-terminal P.69 Prn epitope could be used to assess the latter at a clonal T-cell level. Finally, the P.69 Prn N terminus seems to have an interesting dual role in the adaptive immune response. Earlier, Hijnen et al. found that more than 50% of P.69 Prn-specific antibodies in human sera were directed toward N-terminal epitopes (21) and that N-terminal B-cell epitopes (or epitopes that are dependent on the presence of the N terminus) induce protective antibodies (M. Hijnen et al., unpublished data). Our data now
indicate the involvement of the N terminus as a CD4⫹ T-cell target as well. Topographic linkage between B- and T-helper cell epitopes on the same protein antigen might be functional for the outcome of antibody responses (41, 42, 49). Notably, removal of the N terminus of P.69 Prn was preliminarily found to reduce its protective capacity in mice (Hijnen et al., unpublished). Although the role of the P.69 Prn N-terminus-specific CD4⫹ T-cell response in protection was not addressed in this study, our data indirectly suggest that the observation by Hijnen et al. could be attributed not only to the removal of important B-cell epitopes but also to that of the immunodominant CD4⫹ T-cell epitope. In conclusion, our study sheds new light on P.69 Prn as a target in cell-mediated pertussis immunity, in particular by mapping a cross-species shared CD4⫹ T-cell epitope to a region of P.69 Prn with important immunogenic properties. The unraveling of pertussis-specific immune mechanisms in both experimental and clinical models is much needed in view of the resurgence of pertussis and the current call for improved vaccines. ACKNOWLEDGMENTS We thank H. F. Brugghe and J. A. M. Timmermans for peptide synthesis and D. Canaday for kindly providing the BW1100 cell line. REFERENCES 1. Ad Hoc Group for the Study of Pertussis Vaccines. 1988. Placebo-controlled trial of two acellular pertussis vaccines in Sweden—protective efficacy and adverse events. Lancet i:955–960.
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