Complex Class II-Presented Epitope Repertoire in Human
Monocyte-Derived Dendritic Cells
Rachel M. Stenger,aHugo D. Meiring,bBetsy Kuipers,aMartien Poelen,aJacqueline A. M. van Gaans-van den Brink,a Claire J. P. Boog,bAd P. J. M. de Jong,bCécile A. C. M. van Elsa
Centre for Immunology of Infectious Diseases and Vaccines, National Institute for Public Health and the Environment, Bilthoven, the Netherlandsa; Institute for
Translational Vaccinology, Bilthoven, the Netherlandsb
Knowledge of naturally processed Bordetella pertussis-specific T cell epitopes may help to increase our understanding of the
ba-sis of cell-mediated immune mechanisms to control this reemerging pathogen. Here, we elucidate for the first time the dominant
major histocompatibility complex (MHC) class II-presented B. pertussis CD4
ⴙT cell epitopes, expressed on human
monocyte-derived dendritic cells (MDDC) after the processing of whole bacterial cells by use of a platform of immunoproteomics
technol-ogy. Pertussis epitopes identified in the context of HLA-DR molecules were derived from two envelope proteins, i.e., putative
periplasmic protein (PPP) and putative peptidoglycan-associated lipoprotein (PAL), and from two cytosolic proteins, i.e.,
10-kDa chaperonin groES protein (groES) and adenylosuccinate synthetase (ASS). No epitopes were detectable from known
viru-lence factors. CD4
ⴙT cell responsiveness in healthy adults against peptide pools representing epitope regions or full proteins
confirmed the immunogenicity of PAL, PPP, groES, and ASS. Elevated lymphoproliferative activity to PPP, groES, and ASS in
subjects within a year after the diagnosis of symptomatic pertussis suggested immunogenic exposure to these proteins during
clinical infection. The PAL-, PPP-, groES-, and ASS-specific responses were associated with secretion of functional Th1 (tumor
necrosis factor alpha [TNF-
␣] and gamma interferon [IFN-␥]) and Th2 (interleukin 5 [IL-5] and IL-13) cytokines. Relative
pau-city in the natural B. pertussis epitope display of MDDC, not dominated by epitopes from known protective antigens, can
inter-fere with the effectiveness of immune recognition of B. pertussis. A more complete understanding of hallmarks in B.
pertussis-specific immunity may advance the design of novel immunological assays and prevention strategies.
P
revention of morbidity and mortality caused by the human
pathogen B. pertussis has effectively relied on national
vacci-nation programs since these were introduced in the 1940s and
1950s (1,
2). However, during the last decade an increase of
whooping cough has been reported (3–9). Pertussis, mostly feared
for affecting infants too young to be fully vaccinated, is more and
more notified among adolescents and adults, who apparently
gradually lose their vaccine-induced protection to current B.
per-tussis strains (
2,
10–13). Understanding of protective adaptive
im-munity and its weaknesses is essential for us to be able to improve
pertussis vaccination. Despite being implicated in protection
against severe pertussis (14,
15), levels of preexposure human
se-rum antibodies to the major vaccine antigens have never been
exclusively correlated with the efficacy of pertussis vaccines (16–
18). In addition to antibodies, CD4
⫹T cells contribute to
immu-nological resistance against B. pertussis infection. First, CD4
⫹T
cell responses are essential for avidity maturation of specific B cell
responses. Second, murine (19–25) and human (26–33) B.
pertus-sis-specific CD4
⫹T cell immunity has been associated with the
secretion of Th1-, Th2-, and Th17-type cytokines capable of
arm-ing different types of antimicrobial effector mechanisms, such as
the activities of macrophages, eosinophils, and neutrophils,
re-spectively (34). Unfortunately, information on the fine-specificity
and breadth of human B. pertussis-specific CD4
⫹T cell responses
is largely absent, not in the least because of the difficulty of
prop-agating B. pertussis-specific CD4
⫹T cells in vitro (R. M. Stenger,
M. Poelen, and C. A. C. M. van Els, unpublished data). The few
human B. pertussis-specific CD4
⫹T cell epitopes that have been
elucidated were all sought only on the basis of known virulence
factors (29,
35–37). We were interested in gaining nonbiased
in-sight into the natural repertoire of B. pertussis-specific major
his-tocompatibility complex (MHC) class II-presented epitopes,
available for T cell recognition at the cell surface of human
pro-fessional antigen-presenting cells after processing of the whole
bacterial proteome. We used a dedicated immunoproteomics
method to identify naturally processed and HLA-DR-associated
B. pertussis epitopes isolated from human monocyte-derived
den-dritic cells (MDDC). Stable isotope labeling of B. pertussis biomass
prior to loading on MDDC and nanoscale liquid chromatography
electrospray ionization mass spectrometry (NanoLC-ESI-MS)
were used to sensitively detect pertussis peptide epitopes among
thousands of self-peptides in complex peptide fractions (38). This
approach led to the identification of four B. pertussis proteins as
sources for dominantly selected and functional CD4
⫹T cell target
epitopes. Interestingly, none of the source proteins was a known
virulence factor currently used in acellular pertussis vaccines.
Understanding peculiarities in the display of the natural B.
Received 26 October 2013 Returned for modification 29 November 2013 Accepted 20 February 2014
Published ahead of print 5 March 2014 Editor: T. S. Alexander
Address correspondence to Cécile A. C. M. van Els, [email protected]. Copyright © 2014, American Society for Microbiology. All Rights Reserved.
doi:10.1128/CVI.00665-13
pertussis CD4
⫹T cell epitope repertoire on MDDC, such as strong
epitope domination, may help to elucidate weaknesses in the
hu-man cellular immune response to pertussis and may provide leads
on how to design new generations of pertussis assays and vaccines.
MATERIALS AND METHODS
Subjects and ethics statement. Blood from volunteers was obtained in
accordance with the Declaration of Helsinki and with Dutch regulations, following approval, respectively, from the Sanquin Ethical Advisory Board (for citrated buffy coat donation from 20 HLA-typed healthy blood bank donors and for one leukapheresis donation from an HLA-typed healthy blood bank donor [trial BS03.0015-x]) and from the accredited Review Board STEG and the review boards of collaborating hospitals for heparinized blood sampling of participants from a clinical study (age range, 8 to 77 years [median, 40 years]) (30 pertussis patients within 12 months after laboratory-confirmed diagnosis of B. pertussis infection and 10 healthy household contacts negative for B. pertussis infection based on diagnostic serology [trial NVI-243, CCMO number NL16334.040.07]). All participants provided written informed consent for the collection of samples and subsequent analysis.
Isolation of PBMC. Peripheral blood mononuclear cells (PBMC)
from citrated blood samples were separated by centrifugation on a Ficoll-Hypaque gradient (Pharmacia Biotech, Uppsala Sweden) or on a gel in heparinized CPT tubes (BD Biosciences). After washing and counting, cells were used directly or after cryopreservation.
Bacterial strains and growth conditions. The B. pertussis vaccine
strain 509 was grown on Bordet-Gengou (BG) agar plates containing 15% defibrinated sheep blood. Liquid B. pertussis cultures were grown in either natural14N-containing minimal Bioexpress cell growth medium or in 98-atom%-enriched15N stable isotope containing minimal Bioexpress
cell growth medium (Cambridge Isotope Laboratories) (both containing 0.15% lactic acid [Fluka, Switzerland] adjusted to pH 7.2 with NaOH) until reaching stationary phase and expressing virulence antigens. At this stage, cultures containing B. pertussis whole cells were heat inactivated for 30 min at 56°C and concentrated 5 times in phosphate-buffered saline (PBS) by centrifugation at 2,000⫻ g for 20 min. Before use, heat-inacti-vated14N and15N bacterial biomasses were subjected to quality control.
Nanoscale liquid chromatography electrospray ionization tandem mass spectrometry (LC-MS/MS) sequence analysis of trypsin-digested single SDS-PAGE bands of the14N and15N biomass confirmed uniform stable
isotope labeling of pertussis proteins. The optical densities of both whole-cell biomasses were measured at 600 nm (A600), and based on these A600
values, a 1:1 mixture of the14N- and15N-concentrated whole-cell
bio-masses was prepared for antigen pulsing of human MDDC (step A in the immunoproteomics strategy,Fig. 1A).
Proteins and synthetic peptides. Recombinant P.69 pertactin (P.69
Prn) was expressed and purified from an Escherichia coli construct as previously described (39). Pertussis toxin (Ptx), filamentous hemaggluti-nin (FHA), and fimbriae subtypes 2 and 3 (Fim2/3) were purified in-house according to procedures described in the literature (40–42). Purity of all antigens was confirmed by SDS-PAGE.
Synthetic peptides representing the exact HLA-DR eluted peptide se-quences, as well as 12 amino acid (aa)-overlapping 18-mer peptides rep-resenting the entire protein sequences of 10-kDa chaperonin groES pro-tein (groES) (B. pertussis gene identification number BP3496 [n⫽ 13 peptides]), putative periplasmic protein (PPP) (BP3341 [n⫽ 37 pep-tides]), adenylosuccinate synthetase (ASS) (BP2188 [n⫽ 71 peptides]), and putative peptidoglycan-associated lipoprotein (PAL) (BP3342 [n⫽ 25 peptides]), respectively, were prepared by solid-phase synthesis using N␣-(9-fluorenylmethoxy carbonyl) (FMOC)-protected amino acids and a SYRO II simultaneous multiple peptide synthesizer (MultiSyntech GmbH, Witten, Germany). The purity and identity of the synthesized peptides were assessed by reverse-phase high-performance liquid chro-matography and mass spectrometry. Peptides were used as single
pep-tides, combined adjacent peptides representing a broadened epitope re-gion, or peptide pools representing an entire protein.
Immunoblotting. For evaluation of the relative protein contents of
known virulence factors,14N and15N B. pertussis whole-cell preparations
were run on SDS-PAGE gels (Pierce, Switzerland) and transferred to ni-trocellulose membranes (Schleicher & Schuell). Membranes were washed with Tris/NaCl buffer (pH 7.4) with 0.5% Tween 80 (WS-T), and then probed for 1 h at room temperature (RT) in WS-T with the mouse mono-clonal antibodies anti-FHA (31E2), anti-P.69 Prn (Pem4), anti-Ptx S1 (151C1), and anti-Fim2 (136A6) (all from the Dutch National Institute for Public Health and the Environment [43,44]). Thereafter, the mem-branes were washed and incubated for 1 h in WS-T containing alkaline phosphatase-labeled goat-anti-mouse IgG (Southern Biotechnology As-sociates, Inc.) and 0.5% Protifar (Nutricia, the Netherlands), respectively. Signal was detected with the ready-to-use alkaline phosphatase (AP) con-jugate substrate kit (Bio-Rad).
Culturing, antigen pulse, and characterization of MDDC. Immature
human MDDC were cultured based on a procedure described by Sallusto et al. (34). Briefly, 1⫻ 109PBMC obtained from an
HLA-DR2-homozy-gous blood donor after leukapheresis were seeded at⬃5 ⫻ 106/ml in
150-mm tissue culture dishes (Corning Costar) in Iscove’s modified Dul-becco’s medium (GibcoBRL) supplemented with 1% fetal bovine serum (HyClone) and penicillin-streptomycin-glutamine (GibcoBRL) at 37°C and 5% CO2in a humidified incubator for 2 h. After removal of the
nonadherent fraction, adherent cells were further cultured for 6 days in medium containing 500 U/ml recombinant human granulocyte-macro-phage colony-stimulating factor (GM-CSF) (PeproTech) and 250 U/ml recombinant human IL-4 (Strathmann Biotech GmbH, Germany). Cul-ture medium and growth factors were refreshed on day 3. On day 6, 1.2⫻ 108MDDC, still immature, were pulsed with a 1:1 wt/wt mixture of14 N-and15N-concentrated bacterial biomass at an optimized final
concentra-tion/A600of 0.028 and incubated for 6 h. Thereafter, cells were further cultured and maturated in the continuous presence of antigen, growth factors, and 20 ng/ml lipopolysaccharide (Salmonella abortus equi; Sigma). On day 8, B. pertussis-pulsed and maturated MDDC were har-vested and washed four times in PBS, counted, pelleted, and snap frozen before cell lysis and peptide isolation (Fig. 1B). Maturation of the MDDC was verified using flow cytometry by comparing the expression of CD83, CD40, CD80, and CD86 cell surface markers on day 6 and day 8 MDDC (data not shown).
Isolation and NanoLC-ESI-MS analysis of HLA-DR-associated pep-tides. B. pertussis-pulsed MDDC were lysed and HLA-DR-peptide
com-plexes were isolated essentially as described previously (38), using the HLA-DR-specific monoclonal antibody B8.11.2 bound to CNBr-acti-vated Sepharose 4B beads. Peptides were eluted using 10% acetic acid and spun through a 10-kDa cutoff Spinfilter (Millipore, USA) (Fig. 1C). The filtrate was concentrated to⬃10 l using a freeze dryer and prefraction-ated using strong cation exchange (SCX) chromatography. Peptide frac-tions were reconstituted in a 5% formic acid and 5% dimethyl sulfoxide solution in water containing angiotensin-III and oxytocin (each at a con-centration of 250 amol/l) as internal standard peptides and analyzed using NanoLC-ESI-MS/MS, as described earlier (45) (Fig. 1D). Charac-teristic heavy and light mass spectral doublets, representing MHC class II-associated peptide epitopes originating from corresponding14N and 15N B. pertussis protein homologues, were allocated in all mass spectra
analogue compared to these two standard peptides when acquired under identical NanoLC-ESI-MS conditions.
Functional assays to analyze immunogenicity of epitopes.
Immuno-genicity of epitopes was tested by incubating PBMC in the absence or presence of the indicated synthetic peptide or peptide pool (Fig. 1G) (at 1 M per peptide) in complete AIM-V medium (AIM-V medium contain-ing streptomycin, gentamicin, and L-glutamine [GibcoBRL] supple-mented with 2% human AB serum [Harlan]) at 105cells per well in 150l
in 96-well round-bottom plates (Greiner) for 6 or 10 days in 3-, 10-, or 40-fold replicated wells per condition, as indicated, at 37°C in a humidi-fied 5% CO2atmosphere. Plates were visually inspected and either
super-natants were harvested for cytokine measurement, 0.5Ci (18.5 kBq) [3H]thymidine (Amersham, USA) was added overnight to measure direct
proliferation, or cultures were expanded on IL-2 and restimulated, as described below, before the addition of [3H]thymidine. Eighteen hours
after labeling, cells were harvested and the [3H]thymidine incorporation FIG 1 Immunoproteomics strategy to identify pathogen derived MHC class II-associated peptides. (A) Separate B. pertussis (Bp) cultures are prepared in
was determined as counts per minute (CPM) using a Wallac 1205 beta-plate liquid scintillation counter. Results are expressed as stimulation in-dex (SI) from triplicate (donors from the NVI-243 study) or decuple wells (healthy blood donors), calculated as follows: average cpm of PBMC in the presence of peptide(s)/average cpm of PBMC in medium only (direct proliferation) or average cpm of stimulated PBMC cultures in the pres-ence of peptide-pulsed antigen-presenting cells/average cpm of stimu-lated PBMC cultures in the presence of mock-pulsed antigen-presenting cells (indirect proliferation). SI values ofⱖ1.5 were considered positive.
For cytokine analysis, human Th1/Th2 and Th17 cytokine Bio-Plex kits (Bio-Rad) were used to determine concentrations of IL-2, IL-4, IL-5, IL-10, IL-12(p70), IL-13, IL-17, tumor necrosis factor alpha (TNF-␣), and gamma interferon (IFN-␥) according to the manufacturer’s instruc-tions in (pooled) supernatants taken, as indicated, from those wells out of 40 plated per specific synthetic peptide pool that were visibly activated.
FIG 2 Confirmation of the presence of four virulence factors in the14N- and 15N-labeled B. pertussis whole-cell vaccines. Immunoblots of SDS-PAGE-sep-arated antigen preparations using specific monoclonal antibodies with indi-cated antigen specificities. Lane 1, purified antigen. Lane 2, B. pertussis cultured on Bordet Gengou plates. Lane 3,14N-labeled B. pertussis culture. Lane 4, 15N-labeled B. pertussis culture. FHA, filamentous hemagglutinin; P.69 Prn, P.69 pertactin; Ptx, pertussis toxin; Fim2, fimbriae type 2. The position of molecular mass (kDa) of protein markers is indicated.
Measurements and data analysis were performed with the Bio-Plex system in combination with Bio-Plex manager software. Results are expressed in pg/ml.
For analysis of cross-reactivity, PBMC from a groES34 –52-reactive
healthy donor were stimulated using groES34 –52synthetic peptide for 10
days and then further expanded on 10 ng/ml IL-2 and fresh medium. On day 24, expanded T cells were restimulated for 2 days in triplicate wells using medium only, synthetic peptide representing B. pertussis groES34 –52,
or synthetic peptide representing Homo sapiens hsp1040 –58in the presence
of autologous antigen-presenting cells. Then [3H]thymidine was added
and incorporation was measured after overnight incubation, as indicated above. Results are expressed as stimulation index (SI) from triplicate wells.
Statistics. The statistical significance of differences in ex vivo
lym-phoproliferation between groups was calculated with the Student’s t test (two-tailed, unequal variance).
RESULTS
Immunoproteomics strategy for sensitive detection of MHC
class presented B. pertussis epitopes. Foreign MHC class
II-presented epitopes can be isolated from antigen-presenting cells for
NanoLC-ESI-MS analysis, but to distinguish them from thousands of
self-derived epitopes in complex peptide mixtures is technically
highly challenging. To rapidly recognize B. pertussis-specific peptide
masses in MHC class II eluates, an immunoproteomics strategy
was applied, based on metabolic labeling of the bacterial proteome
(schematically illustrated in
Fig. 1A
to
F). Antigenically, the
14N
and
15N whole-cell biomasses, harvested in the virulence (bvg
⫹)
phase, were comparable, as was confirmed by immunoblotting of
the known virulence factors P.69 pertactin (P.69 Prn), pertussis
toxin subunit 1 (PtxS1), filamentous hemagglutinin (FHA), and
fimbriae 2 (Fim2) (Fig. 2). After processing of 1:1 wt/wt mixed
bacterial biomasses by MDDC, an HLA-DR-derived peptide
sam-ple was obtained, fractionated, and analyzed for the presence of
candidate B. pertussis-derived epitopes (Fig. 1D
to
F).
B. pertussis PAL, groES, PPP, and ASS identified as dominant
source proteins of naturally processed and MHC class
II-pre-sented epitopes. An example of such a candidate epitope is given
in
Fig. 3A
and
B. Epitope sequencing by targeted nanoscale
LC-MS/MS and database matching indeed identified its source
pro-tein as a B. pertussis propro-tein, groES (Fig. 3C). In total, seven
natu-rally processed and presented and hitherto unknown peptides,
representing epitopes from four different B. pertussis proteins,
in-cluding length variants, were identified using isotope-tagged
iden-tification (Table 1). A peptide representing aa residues 103 to 118
from the envelope PAL protein, PAL
103–118, was the most
abun-dant epitope, with an estimated MHC-peptide copy number of
350 epitopes per MDDC. Lower epitope densities were found for a
natural length variant of this epitope and epitopes from groES,
PPP, and ASS. No mass spectral doublets were assigned to known
B. pertussis virulence factors, despite the fact that these proteins
were present in the B. pertussis biomass preparations that were
used for MDDC loading (Fig. 2). Also, data-dependent mass
se-quencing of peptides present in the eluate did reveal the presence
of a wide repertoire of HLA-DR-associated self-sequences (data
not shown), but this approach did not yield any additional B.
pertussis epitopes.
Naturally processed and HLA-DR-presented epitope regions
represent functional CD4
ⴙT cell targets. To test the
immunoge-nicity of the processed and HLA-DR2-presented pertussis
pep-tides, PBMC from an HLA-DR-matched blood donor were
stim-ulated ex vivo using synthetic peptides representing the eluted
groES
34 –52, PPP
132–146, ASS
166 –179, and PAL
103–118sequences. In
this donor, direct lymphoproliferation could be measured against
all four epitopes (Fig. 4).
We reasoned that the identified peptide sequences may not
only represent the exact HLA-DR2-presented epitopes, but may
also indicate a broader protein region liberated by proteolysis and
possibly containing multiple HLA-DR-binding motifs. We also
assumed, since the epitopes were in vitro derived from an
experi-mental whole-cell vaccine, that childhood whole-cell pertussis
vaccination could be sufficient to prime CD4
⫹T cell responses to
these epitopes. To evaluate this, PBMC from 20 healthy
HLA-DR2-typed adults from a birth cohort associated with whole-cell
pertussis vaccination were stimulated with pools of overlapping
synthetic peptides representing the epitope regions, including
flanking sequences, to encompass adjacent motifs that were
pre-dicted to bind a wide array of HLA-DR molecules (46,
47) (data
not shown). As summarized in
Fig. 5, direct specific proliferation
was observed against all four epitope regions with 30 to 40%
over-TABLE 1 Naturally processed and HLA-DR2-presented epitopes of B.
pertussis whole-cell biomass
Source protein Epitope Abundancea
Chaperonin groES protein (groES) (BP3496b ) 34 KPDQGEVVAVGPGKKTED51 5 34 KPDQGEVVAVGPGKKTEDG52 80 Putative periplasmic protein (PPP) (BP3341) 135IALYPNSQLAPT146 30 132 AAFIALYONSQLAPT146 175 Adenylosuccinate synthetase (ASS) (BP2188) 166LAEVLDYHNFVLTQ179 10 Putative peptidoglycan-associated lipoprotein (PAL) (BP3342) 103 GGAEYNLALGQRRA116 10 103GGAEYNLALGQRRADA118 350 aCopies/cell. b
B. pertussis gene identification number (48).
all responsiveness, not limited to HLA-DR2
⫹donors only. These
data suggest that the extended epitope regions of groES, PPP, ASS,
and PAL are recognized in a broader immunogenetic context.
To investigate the type of helper T cell responses associated
with epitope immunogenicity, PBMC from responding donors 1,
5, 14, and 19 were cocultured in a new set of restimulations with
relevant sets of peptides and cytokine analysis on culture
superna-tants of wells with activated cultures. All epitope regions were
associated with both Th1- (IFN-
␥ and TNF-␣) and Th2-type
(IL-5 and IL-13) cytokine responsiveness (Fig. 6). No other
cyto-kines, such as IL-10 or IL-17, were detected.
Specific proliferation as well as cytokine production of
ex-panded CD4
⫹T cell cultures from various donors could be
blocked by monoclonal antibodies against HLA-DR molecules,
confirming the involvement of HLA-DR as a restriction element
(data not shown). Collectively, these findings indicate that the
four HLA-DR2-eluted epitopes represent favorably processed and
presented protein regions immunogenic for CD4
⫹T cells and
have a mixed Th1/Th2 cytokine signature.
Epitope-specific responses observed after clinical pertussis
infection. To test whether epitopes from groES, PPP, ASS, and
PAL were naturally exposed during infection, PBMC obtained
from pertussis patients within 12 months after their
laboratory-confirmed clinical episode (n
⫽ 30) and from healthy controls
(n
⫽ 30) were stimulated with overlapping synthetic 18-mer
pep-tides covering the full protein sequences and were assayed for
proliferative responses. As illustrated in
Fig. 7, a higher percentage
of patients had proliferative responses above the SI cutoff of 1.5
than did healthy controls when tested against peptide sets of
groES, PPP, and ASS, but not of PAL. When tested statistically,
differences in lymphoproliferation between groups were not
sig-nificant. The trend, however, may implicate a boosting effect of
infection for CD4
⫹T cell responses of groES, PPP, and ASS.
groES
34 –52-specific CD4
ⴙT cells do not cross-react to the
ho-mologous human hsp10 epitope. The 10-kDa chaperonin groES
protein is a family member of the low molecular weight heat shock
proteins, which are conserved not only between bacteria, but also
between humans and other mammal species. The identified B.
pertussis groES epitope shows 52.5% sequence homology with the
counterpart region on Homo sapiens hsp10 (Fig. 8). To investigate
whether this homology could be immunologically relevant or
even underlie autoimmunity, we compared the capacity of
groES
34 –52and its human homologue hsp10
40 –58to restimulate
an in vitro-expanded B. pertussis groES
34 –52-specific CD4
⫹T cell
bulk culture in the presence of autologous antigen-presenting
cells. As shown in
Fig. 9, no recognition of the H. sapiens hsp10
epitope was observed, suggesting that priming of human T cells
with the dominantly presented B. pertussis groES region is unlikely
to result in cross-reactivity against endogenous tissue.
DISCUSSION
Using an unconventional immunoproteomics approach, we
iden-tified hitherto unknown naturally processed and dominantly
pre-sented B. pertussis-specific CD4
⫹T cell epitopes. This approach
has two major advantages over strategies aimed at mapping
epitopes on predefined protein targets. First, discovery of epitopes
is made independent of their protein source, which is especially
relevant for pathogens with large proteomes, such as B. pertussis,
with more than 3,800 open reading frames (48). Second, relative
abundances and natural length variants of epitopes can be
deter-mined, which may have an immunological impact.
The B. pertussis epitopes discovered in this study to be MHC
class II presented and immunogenic in humans originated from
four different bacterial source proteins localizing to two different
subcellular compartments of B. pertussis whole cells. PPP
(alter-native name, YbgF) and PAL, encoded by adjacent genes on the
same operon, are both components of the cell envelope Tol-Pal
system, critical for the integrity of the bacterial outer membrane
(49). The 10-kDa chaperonin groES, involved in protein folding
and assembly as a homoheptameric ring associated with the
chap-eronin cpn60, and ASS, catalyzing the first step in the de novo
biosynthesis of AMP, are both located in the cytoplasm (48).
Unexpectedly, none of the dominantly presented epitopes
originated from known B. pertussis virulence factors, such as FHA,
P.69 Prn, PtxS1, or Fim2, while these proteins were abundantly
present in the digested bacterial biomass (Fig. 2) and some encode
r o n o d 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
groES
19-54PPP
133-162ASS
163-192PAL
103-138FIG 5 Ex vivo lymphoproliferation to peptides representing naturally MHC class II-presented B. pertussis epitope regions in a panel of healthy HLA-typed
donors. N- and C-terminal amino acid positions of protein region are represented by 12-mer overlapping 18-mer synthetic peptides. HLA-DR2⫹donors, 1, 11, 16, and 17; HLA-DR2⫺donors, 2 to 9, 12 to 15, and 18 to 20; untyped donor, 10. White cells, mean SI (of decuple wells per stimulation) of⬍1.4; light-gray cells, mean SI ofⱖ1.4 and ⱕ3.0; dark-gray cells, mean SI of ⬎3.0.
groES19-54 Donor PPP133-162 Donor
1 5 14 1 5 14
Responsiveness 16 21 1 Responsiveness 8 5 1
Th1 IFN-γTNF-α Th1 TNF-α IFN-γ
Th2 IL-13 IL-5 Th2 IL-13 IL-5
ASS163-192 Donor PAL103-138 Donor 9 1 4 1 5 1 9 1 5 1 Responsiveness 10 9 2 Responsiveness 1 8 2 5 Th1 IFN-γ Th1 IFN-γ TNF-α TNF-α Th2 IL-5 Th2 IL-5 IL-13 IL-13
FIG 6 Cytokines associated with responses against naturally MHC class
known T cell epitopes (29,
35,
50,
51). In fact, preceding B.
pertus-sis harvesting, in-culture gene expression values for groES, PPP,
ASS, and PAL were comparably high (groES and PAL) or even
lower (PPP and ASS) than those for FHA, P.69 Prn, PtxS1, or Fim2
(52) (B. van der Waterbeemd, personal communication).
Therefore, a high antigen load may be favorable, but it is not
sufficient for a source protein to dominate in the MHC class II
ligandome of MDDC. It is known that for epitopes to be formed,
modest but not destructive proteolysis of source proteins (53) and
peptide affinity to MHC class II molecules (54) are prerequisites.
The groES, PPP, ASS, and epitope regions all have very high
pre-dicted epitope binding scores for either HLA-DRB1*1501 or
HLA-DRB5*0101, the two HLA-DR molecules expressed on the
HLA-DR2
⫹MDDC (data not shown). Hence, they can be
as-sumed to be strong competitors for binding with epitopes derived
from other proteins. Also, other features driving exclusive
presen-tation by MDDC could be involved, such as those described for
the Toxoplasma gondii protein profilin, being immunodominant
in the CD4
⫹T cell response to the pathogen solely because of
enhanced and selective TLR11-mediated uptake (55). TLR2 could
play a role in the uptake of the lipoprotein PAL (56–62), possibly
in association with the other Tol-Pal component PPP. TLR2
might also sense groES, eventually via associated compounds (63–
65). It is unknown whether ASS has innate receptor affinity.
Another unexpected observation was the modest average
epitope abundance of the pertussis MHC class II ligands, with
estimated numbers of MHC-peptide complexes per cell of 5 to
350. This was dissimilar to the range of MHC class II-presented
epitopes we described previously, using an MDDC model with
meningococcal outer membrane vesicles as the antigen and
apply-ing the same degree of sample prefractionation and analytical
sen-sitivity. HLA-DR-associated meningococcal epitopes isolated
from either HLA-DR1 homozygous or HLA-DR2 homozygous
MDDC ranged from 30 to 10,000 copies per cell (reference
38
and
FIG 7 Clinical infection enhances CD4⫹T cell lymphoproliferation to different B. pertussis epitope regions. PBMC of pertussis patients within 12 months after laboratory-confirmed pertussis (n⫽ 30) and of healthy controls (n ⫽ 10 noninfected household contacts and n ⫽ 20 healthy blood bank donors) were stimulated with pools of 18-mer peptides, representing the entire indicated proteins at 1M per peptide. [3H]Thymidine incorporation was determined 7 days after in vitro stimulation. Dots represent SI from triplicate wells of different individuals.
Species Amino acid sequence
B. pertussis groES 25IVIPDSAAEKPDQGEVVAVGPGKKTEDGKILPVDLK 60
H. sapiens hsp10 31IMLPEKSQGKVLQATVVAVGSGSKGKGGQPVSVKVG 66
FIG 8 Sequence homology between B. pertussis and H. sapiens 10-kDa chaperonins. Gray letters indicate the amino acid sequence of the longest naturally
H. Meiring, unpublished data). Although we cannot exclude a
certain degree of peptide loss, potentially also from unidentified
epitopes during the sample isolation procedure, or of
undersam-pling of MHC ligand identification at the mass spectrometry level
(66), it is unlikely that this fully accounts for the large difference
between the pertussis and the meningococcal epitope repertoire.
MDDC pulsed with B. pertussis whole-cell vaccine had
compara-ble levels of HLA-DR molecules at the cell surface, of MHC
pro-tein retentate, and of self-peptides in the eluate as did
meningo-coccal outer membrane vesicle maturated MDDC (data not
shown). Hence, our data indicate a selective and limited breadth
and density of B. pertussis MHC class II epitopes on human
MDDC, among which additional epitopes from groES, PPP, ASS,
and PAL or epitopes from virulence factors were either absent or
too low in copy number to be detected by our system. Limited
epitope display, especially from virulence factors, could
circum-vent effective immune recognition and mechanisms crucial for
protection. While it has been shown that B. pertussis antigens can
interfere with MDDC maturation and function (67), low and
highly selective epitope expression on MDDC might be another
successful feature of B. pertussis to evade the adaptive immune
response. Further immunoproteomics research, extending MHC
class II ligandome analyses to other HLA-DR alleles,
antigen-pre-senting cell types, and antigen preparations and to experimental
infection, is needed to substantiate this hypothesis.
The roles of groES, PPP, ASS, and PAL antigens as targets in
host immunity remain to be elucidated. A direct protective role as
targets for CD4
⫹T cell-dependent antibody responses, which
could be tested in an animal challenge model, is not expected,
since the antigens are not surface exposed on the bacterium.
How-ever, the antigens could otherwise serve as relevant targets of
im-mune mechanisms. The induced specific CD4
⫹T cell populations
could enhance pertussis immunity via the generation of an
in-flammatory cytokine milieu, or alternatively, could dampen other
CD4
⫹T cell specificities through competition for space (68) or
through regulation. The CD4
⫹T cell responses to the identified
epitope regions, observed in a considerable proportion of tested
individuals, were associated with a mixed Th1/Th2 cytokine
pro-file, i.e., secretion of IFN-
␥, TNF-␣, and IL-5 and IL-13, in the
absence of IL-10 and IL-17. IFN-␥ and TNF-␣ are both
inflam-matory cytokines that have been implicated in controlling B.
per-tussis through potentiating bactericidal activity of macrophages
and enhancing phagocytosis by neutrophils (69–71), suggesting a
role in protection. Notably, (ex)patients had slightly elevated
liferative responses to three of four naturally processed target
pro-teins, possibly indicating that the epitopes were exposed during
infection and could play a role in natural CD4
⫹T cell immunity to
B. pertussis. While the data set was possibly too limited to be
con-clusive and testing more patients’ samples could help to settle this
point, it is also possible that elevation of T cell responses is
con-fined to a very narrow time span after boosting. The majority of
patients in this study were adults who were within 2 months after
their formal diagnosis. However, since B. pertussis has a relatively
long incubation time (14 to 21 days) and adults are often
diag-nosed only after a period of prolonged coughing, an early boosting
effect of the epitopes on T cell levels could have easily been missed.
Responses to the B. pertussis groES epitope were
non-cross-reactive to the endogenous human hsp10 homologue. Therefore,
dissimilar to the CD4
⫹T cell epitope cross-reactivities observed
for some mycobacterial and human hsp70 protein families, a
po-tential role in autoimmunity or immunoregulation for B. pertussis
groES is not envisaged (64).
In conclusion, an unexpected limited epitope breadth and
abundance of human MDDC-presented B. pertussis-specific
CD4
⫹T cell epitopes was found, with a role for other proteins
than those that are known virulence factors. Clearly, although
being a nonroutine and highly technical approach,
immunopro-teomics can shed light on classes of otherwise elusive T cell
epitopes. These results represent a step toward a more complete
characterization of the natural immune response to B. pertussis,
involving other specificities than hitherto anticipated. Ultimately,
a better understanding of the immunological correlates of
protec-tion against whooping cough and their flaws is needed to develop
new functional assays and more effective vaccines.
ACKNOWLEDGMENTS
We are grateful to the volunteers and personnel from Sanquin Blood Bank North West for blood donations (S03.0015-x) and to volunteers and em-ployees from the SKI-study (NVI-243). We thank P. Hoogerhout, H. F. Brugghe, and J. A. M. Timmermans for peptide synthesis, J. A. M. Tim-mermans for preparing figures, B. van de Waterbeemd and P. van der Ley for discussion, and D. van Baarle for advice.
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