REVIEW

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Investigating pertussis toxin and its

impact on vaccination

Loic Coutte*,1,2,3,4 & Camille Locht1,2,3,4

ABSTRACT Whooping cough, caused by Bordetella pertussis, remains a major global health
problem. Each year around 40 million of pertussis cases resulting in 200,000–400,000 annual
deaths occur worldwide. Pertussis toxin is a major virulence factor of B. pertussis. Murine
studies have shown its importance in bacterial colonization and in immunomodulation to
evade innate or adaptive immunity. The toxin is composed of an A protomer expressing ADP-
ribosyltransferase activity and a B oligomer, responsible for toxin binding to target cells. The
toxin is also a major protective antigen in all currently available vaccines. However, vaccine
escape mutants with altered toxin expression have recently been isolated in countries with
high vaccination coverage illustrating the need for improved pertussis vaccines.

The Gram-negative bacterium Bordetella pertussis is the etiologic agent of whooping cough, also KEYWORDS 
known as pertussis, and is specialized to strictly infect the human respiratory tract. Unvaccinated • A-B toxins • ADP-
neonates and infants constitute the most vulnerable population to develop the severe symptoms of ribosylation • Bordetella
the disease. Typical pertussis symptoms in infants include paroxysmal cough with whooping and pertussis • pathogen
vomiting, associated with pulmonary complications, sometimes leading to death [1] . Older children adaptation • pertussis
and adults are also susceptible, but usually present with milder symptoms, such as general weak- toxin • pertussis vaccines
ness and persistent cough, which can last for 2–4 weeks. Infection in these populations can also • type IV secretion system
be asymptomatic [2] . Although not life-threatening, subclinical pertussis infection in young adults • whooping cough
is of concern, as these subjects constitute an important reservoir for transmission of the infection
to infants, sometimes before infant vaccination has started. Pertussis vaccination with whole-cell
vaccines (wPV) was initiated during the 1940s. Its large-scale implementation in the 1950s and
the 1960s in most industrialized countries has resulted in a rapid decline in the number of cases.
However, in the 1970s, concerns about the reactogenicity of wPV led to reduced compliance and
almost immediate resurgence of the disease [3] . This prompted the development of less reactogenic,
acellular vaccines (aPV). These aPV have now almost completely replaced the wPV in the Western
world. Although initial efficacy trials with aPV indicated protective efficacies comparable with
those of wPV, more recent reports suggest that immunity induced by aPV wanes much faster than
that induced by the wPV [4] . We witness now an important re-emergence of the disease in many
countries. The reasons for this apparent re-emergence are still unclear. They may linked to improved
diagnosis and clinical cases reporting or a decrease in vaccination coverage in some areas. However,
waning of the immune responses to aPV and potentially vaccine-induced genomic changes in
c­irculating strains are also plausible causes [5] .

1
Center for Infection & Immunity of Lille, Institut Pasteur de Lille, 1, rue du Prof. Calmette, F-59019 Lille Cedex, France
2
Inserm U1019, Lille, France
3
CNRS UMR8204, Lille, France
4
Univ Lille Nord de France, Lille, France
*Author for correspondence: Tel.: +33 3 20 87 11 46; Fax: +33 3 20 87 11 58; loic.coutte@inserm.fr part of

10.2217/FMB.14.123 © 2015 Future Medicine Ltd Future Microbiol. (2015) 10(2), 241–254 ISSN 1746-0913 241
Review  Coutte & Locht
Pertussis vaccines & the discovery of
pertussis toxin as a protective antigen
First attempts to develop vaccines against
whooping cough began almost simultane-
ously with the discovery of the causative agent
B. pertussis in 1906 by Bordet and Gengou [6] .
However, it was not until the 1940s that wPV,
combined with diphtheria and tetanus toxoids,
started to be widely used as the diphtheria-
tetanus-pertussis combination vaccines. In
these combinations, the pertussis component
consisted of heat- or formalin-inactivated whole
bacteria, and these vaccines were extremely het-
erogeneous in composition. Nevertheless, the
effectiveness of wPV in reducing global pertus-
sis disease incidence in infants has largely been
demonstrated since the late 1960s (for review,
see [3] ). With the decrease in pertussis inci-
dence and public concern about adverse reac-
tions confidence in wPV diminished, resulting
in decreased vaccination compliance, and in
some countries even to a full vaccination stop,
and the incidence of pertussis increased again.
The development of a less reactogenic pertussis
vaccine became thus a priority.
Attempts to separate the reactogenic compo-
nents of wPV from its protective components
led to the concept of aPV. The first molecules to
be considered as protective antigens and shown
to protect in the mouse potency assays were
hemagglutinins [7] . B. pertussis produces at least
two distinct hemagglutinins. One of them has a
filamentous structure and is named filamentous
hemagglutinin (FHA). The second hemagglu-
tinin has a globular structure and expresses a
number of biological activities, such as hista-
mine sensitization and lymphocytosis promot-
ing activities [8,9] . These and other toxic activi-
ties were later ascribed to a single oligomeric
secreted protein, today referred to as pertussis
toxin (PTX) [10] .
Based on these molecules, aPV were devel-
oped first in Japan, then also in Europe and the
USA. To detoxify PTX, formalin was used, and
these first aPV contained formalin-treated PTX
(PTd) and FHA (FHAd) [8] . PTd was rapidly
identified as the essential antigen in aPV, and
all current aPV contain at least PTd, but the
addition of FHAd appeared to increase the pro-
tection in mouse potency assays. With a substan-
tially improved safety profile of aPV over wPV
and comparable efficacy in Phase III trials, aPV
was well accepted, and the incidence of pertussis
decreased again [3] .
242 Future Microbiol. (2015) 10(2)
Many studies were then undertaken to further
improve aPV. Different detoxification methods
of PTX were tested, the most widely used once
are formaldehyde and glutaraldehyde treatment.
However, due to the lack of lysine residues in
the catalytic S1 subunit, stringent detoxifica-
tion procedures are required resulting in loss of
antigenic properties [11] . With the discovery of
the genes coding for PTX, genetic engineering
became possible to improve aPV. Amino acids
involved in the activity and toxicity of PTX
were found (Figure 1) [12–18] and immunogenic
but nontoxic PTX mutant proteins were con-
structed and tested as new genetically detoxi-
fied PTX antigens in aPV. The most promising
genetically detoxified PTX protein (9K/129G)
was then developed and used in genetically engi-
neered aPV [19,20] . However, this vaccine is not
on the market anymore.
In addition to PTX and FHA, some aPV also
contain the adhesins pertactin (Prn) and fim-
briae (Fim2 and Fim3) [21,22] . Currently, several
aPV are commercially available. They are com-
posed of one (PTX), two (PTX + FHA), three
(PTX, FHA, Prn) or five B. pertussis components
(PTX, FHA, Prn, Fim2, Fim3) [23] . In addition,
aPV antigens are combined with diphtheria and
tetanus toxoids, as well as with inactivated polio
vaccine, Haemophilus influenzae B capsule and
sometimes with the hepatitis B vaccine.
With the exception of one vaccine, all aPV
contain thus multiple B. pertussis antigens. The
monocomponent PTX vaccine was tested in an
efficacy trial in Sweden and was found to pro-
vide protection in children, especially against the
most severe forms of the disease. This vaccine is
currently used in the Danish childhood vaccina-
tion program [24,25] . It has been proposed that
the efficacy of the monocomponent PTX vac-
cine, when given at high dosage, is similar to that
of the multicomponents aPV and that therefore
PTX is both an essential and sufficient antigen
in aPV. In order to improve PTX-based aPV, a
genetically detoxified PTX protein (9K/129G)
has recently been used as a carrier for lipooli-
gosaccharide (LOS) from B. pertussis to induce
both anti-PTX and bactericidal anti-LOS anti-
bodies [26] . However, the protective efficacy in
mouse potency assays or in clinical studies has
not been documented yet.
The structure of PTX
PTX is one of the most complex bacterial pro-
tein toxins. It is a 105-kDa multimeric protein
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composed of five different subunits, named S1
to S5, according to their decreasing molecular
weights. While the genes coding for the toxin
subunits are present in the genomes of Bordetella
parapertussis and Bordetella bronchiseptica, only
B. pertussis produces and secrets the toxin [27] .
In the other two Bordetella species, the ptx genes
are not expressed due to the presence of nucleo-
tide polymorphisms in the promoter regions
[28] . Nevertheless, a clinical isolate of B. bron-
chiseptica has been described to be able to induce
serum antibodies against PTX [29] . However, the
ptx promoter region sequence of this isolate has
not been reported. The lack of PTX production
by B. parapertussis may perhaps explain why
infection with this organism usually results in
milder pertussis-like disease with the absence of
lymphocytosis.
PTX is a member of the ADP-ribosylating
toxin family, which also includes cholera toxin
and diphtheria toxin [30] . It ADP-ribosylates
the α-subunit of the trimeric Gi protein [31] .
The first PTX substrate identified was Giα,
and ADP-ribosylation of Giα by PTX results
in uncoupling of the G protein form its cognate
receptor and thereby inhibits the negative regu-
lation of the cellular adenylate cyclase [32] . Other
G proteins are also substrates of PTX in different
cell types, and their ADP-ribosylation impacts
on various metabolic functions, explaining the
wide variety of biological activities of this toxin.
The enzymatic activity of PTX is carried by
S1, its largest subunit, also referred to as the A
protomer [33] . Subunits S2 to S5 constitute the B
oligomer and are responsible for the binding of
the toxin to its receptors on the surface of the tar-
get cells. As such, PTX is thus a member of the
A-B family of toxins. The PTX crystal structure
shows a pyramid-shaped molecule with the S1
subunit on the top of a triangular base formed by
the B oligomer (Figure 1) [34] . Subunits S2 and S3
form each a dimer with S4 and share significant
sequence similarities [12,35] . Although natural
PTX is arranged in a 1S1:1S2:1S3:2S4:1S5 stoe-
chiometry, mutant strains lacking the S2 gene
can produce toxin homologs containing two S3
subunits. Vice versa, toxin versions lacking S3
can also be assembled and secreted with two
­copies of S2 per molecule [36] .
Biogenesis of PTX
The five PTX subunit structural genes are clus-
tered within a single operon on the B. pertus-
sis chromosome [12,35] . They are followed by 9
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Investigating pertussis toxin & its impact on vaccination  Review
A B
C D
Figure 1. Crystal structure of the pertussis
toxin. (A) Side, (B) opposite side and (C) bottom
side views of the PTX holotoxin. Toxin subunits
are defined by colors: S1 in orange, S2 in dark
blue, S3 in marine blue, S4 in magenta and S5
in cyan. Carbohydrate recognition domains
present in the S2 and S3 subunits are colored in
yellow and sand, respectively. (D) Active site of
the S1 subunit. The amino acids involved in the
activity and toxicity of PTX surrounding the NAD
binding cavity are labeled in blue Arg-9, Trp-26,
His-35, Cys-41 and Glu-129.
additional genes that code for the proteins, called
PtlA to PtlI, involved in PTX secretion. They are
part of the same operon as the structural genes
[37] . The promoter of this operon is under the
control of a two-component regulatory system,
named BvgA/S [38] which regulates the produc-
tion of most of the B. pertussis virulence factors.
The signals that are sensed by the BvgA/S sys-
tem in vivo are not known. After transcription,
the different PTX subunits are synthesized as
preproteins and secreted into the periplasm via
the Sec pathway, where the N-terminal signal
peptides are removed [39] and the subunits are
assembled into the functional holotoxin, even in
the absence of the Ptl secretion machinery [40,41] .
Via the Ptl system, a member of the type
IV secretion system, PTX is finally secreted
through the outer membrane [42] . All Ptl pro-
teins are required for the secretion of PTX
[43] and they form a complex machinery that
spans the entire bacterial envelope. Two of
these proteins, PtlC and PtlH, are cytoplasmic
inner membrane-associated ATPases, which are
responsible for providing the energy required
for secretion [44,45] (for a recent review, see [32]).
The Ptl-specific secretion signals of PTX are
still unknown. However, secretion does not
require the presence of S1, as the assembled B
oligomer can be secreted even in the absence of
S1, indicating that some secretion determinants
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Review  Coutte & Locht
are located on the B oligomer [40] . In the other
hand, alterations in the S1 subunit of PTX affect
its secretion, suggesting that a domain of the
S1 subunit may also carry some secretion deter-
minants required when the holotoxin is fully
assembled [46] .
Toxin binding, trafficking & enzymatic
activities on the host target cells
Although the PTX receptors have not yet been
molecularly defined, virtually all eucaryotic cells
contain a receptor for the toxin. The receptors
are most likely glyco-conjugates, such as sialo-
glycoproteins or glycolipids [47,48] , although
binding to nonglycosylated peptides has also
been reported [49] . The crystal structure of PTX
bound to a undecasaccharide revealed at least
two carbohydrate-binding sites on the S2 and
S3 subunits, respectively (Figure 1) [50] . However,
the two binding sites display a certain degree
of receptor-binding specificities [51] . Surface
plasmon resonance studies using carbohydrate
ligands showed that binding to sialylated com-
pounds is mediated by the C-terminal binding
sites on the S2 and S3 subunits, while binding
to nonsialylated N-linked glycans is mediated by
their N-terminal sites [52] .
Through an as yet unknown mechanism,
binding of PTX to its target cell receptors trig-
gers receptor-mediated endocytosis of the toxin,
followed by retrograde transport involving the
Golgi apparatus and the endoplasmic reticulum
(ER) [53,54] . Dissociation of S1 from the B oli-
gomer most likely occurs in the ER, and after the
dissociation of S1 the B oligomer may undergo
anterograde transport to the Golgi, while S1 is
translocated into the cytosol [55] by a mecha-
nism that has not been investigated yet, but
may involve the ability of S1 to directly bind to
phospholipids bilayers (Figure 2) [56] . Due to the
absence of lysine residues in the S1 subunit the
protein is able to evade the endoplasmic retic-
ulum-associated protein degradation (ERAD)
pathway, thereby allowing it to be released
­efficiently into the cytosol [57,58] .
After translocation S1 remains bound to the
inner leaflet of the ER membrane [53] where it
catalyzes the cleavage of NAD and the transfer
of its ADP-ribose moiety onto the Gα substrate
proteins. This enzyme reaction involves residues
His-35, Trp-26 and Arg-9 for NAD binding, and
Glu-129 for catalysis of the NAD-glycohydrolase
activity (Figure 1D) (see [59] for a review on the
enzyme mechanism).
244 Future Microbiol. (2015) 10(2)
Roles of PTX during infection
A central role of PTX in the pathogenesis of
whooping cough has been suggested since its
discovery, and for decades whooping cough was
considered as a toxin-mediated disease, similar
to tetanus and diphtheria [60] . Specific hallmarks
of clinical pertussis include leukocytosis, hista-
mine sensitization and other toxin-mediated
manifestations, and before the discovery of its
molecular identity PTX was known under dif-
ferent names, such as leukocytosis-promoting
factor, histamine-sensitization factor, islet-acti-
vating protein and hemagglutinin. Once PTX
could be obtained as a pure molecule, all these
activities could be ascribed to a single molecular
entity [10] . It is now well established that most of
these biological activities rely on the S1-mediated
ADP-ribosyltransferase activity. However, some
activities, such as T cell mitogenicity, hemagglu-
tination and others (see below), depend solely on
the B-oligomer binding activities and are inde-
pendent of the enzyme activity of the toxin [51,61] .
The main demonstrated substrates for PTX-
catalyzed ADP-ribosylation are the α subunits
of the Gi/o proteins, but other G proteins can
also be targeted. ADP-ribosylation of Giα locks
the protein in the inactive state and prevents it
from transmitting G-protein receptor-mediated
signals for inhibition of the cellular adenylate
cyclase activity. This results in increased lev-
els of the intracellular concentration of cAMP,
thereby disturbing a variety of cellular meta-
bolic processes, which, depending on the cell
type intoxicated by PTX may account for some
of its biological activities [62] . Other cellular
functions can be disturbed by PTX-catalyzed
ADP-ribosylation of other members of the Gi/o
protein family.
Most experimental infection experiments with
B. pertussis illustrating the role of PTX in infec-
tion made the use of mouse models. Although
mice do not cough, they can be readily infected
by nasal drops or aerosol with B. pertussis and
develop PTX-mediated leukocyosis [8] . Using
these models, PTX release from B. pertussis dur-
ing the infection was shown to be important for
optimal experimental infection, especially at
the early stages of infection. B. pertussis mutant
strains that do not produce PTX show a defect
of colonization within 1–2 days postinoculation
[63] . However, a classical colonization pattern for
this strain can be restored when they are mixed
with a strain that produces and secretes PTX,
or when they are administered together with
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PTX binding
Endosome
Golgi
apparatus
Endoplasmic
reticulum
Nucleus
Future Microbiology © Future Science Group (2015)
Figure 2. Schematic representation of the pertussis toxin trafficking from the extracellular milieu
to the release of the S1 subunit into the cytosol.
purified PTX, indicating that PTX delivered
in trans can restore the colonization of the PTX
nonproducing strains [63] . Even when PTX is
nasally delivered 2 weeks before the administra-
tion of a PTX-deficient mutant, it is still able to
complement the PTX deficiency of the mutant.
However, this is not the case when the toxin is
delivered systemically, or when it is administered
one day after infection with the mutant strain.
While most of the physiological effects of PTX
are related to the systemic features of whooping
cough, such as leukocytosis [60,64] these data thus
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Investigating pertussis toxin & its impact on vaccination  Review
Endocytosis and
endosome trafficking
Retrograde transport from
the Golgi apparatus to the
endoplasmic reticulum
Toxin dissociation and
translocation of S1 into
the cytosol
indicate that PTX may also act locally during
the very first stages of infection on cells already
present in the airways.
Interestingly, the colonization defect of PTX-
deficient mutants is strongly exacerbated when
the strains lack FHA in addition to PTX [65] .
This apparent redundancy depends on the ADP-
ribosyltransferase activity of the toxin and is not
due to a potential role of PTX as an adhesin.
Nevertheless, strains lacking both FHA and
PTX present a defect in their ability to adhere
to human macrophages in vitro, while strains
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Review  Coutte & Locht
lacking either FHA or PTX are not affected in
their adhesion properties [66] .
PTX may also directly or indirectly con-
tribute to the respiratory tract pathology in
the mouse model. Murine infection with
PTX-deficient mutants resulted in substantial
decreased pulmonary inflammation than infec-
tion with PTX-producing isogenic strains, with
a strong reduction in alveolitis, influx of mac-
rophages, lymphocytes and polymorphonuclear
leukocytes in the bronchoalveolar lavage fluids
[67] . More recently, genome-wide transcriptomic
analyses of lungs infected with B. pertussis have
revealed an extensive and prolonged upregula-
tion of inflammatory response genes and genes
implicated in pathology upon infection, which
was not seen when the mice were infected with
a PTX-deficient mutant [68] . Infection with
PTX-deficient mutants only induce an early
and transient inflammatory gene expression
profile, whereas in mice infected with the PTX-
proficient strains, the inflammatory response
continued beyond the peak of B. ­pertussis
infection.
The role of PTX in the pathogenesis of
whooping cough in humans is not fully under-
stood, with exception to its probable role in leu-
kocytosis, one of the general features of human
pertussis. Especially the role of this toxin in
cough remains elusive and cannot be studied
in mouse models, as mice do not cough. More
than 20 years ago, a coughing rat model was
developed and used to test various B. pertussis
mutant strains [69] . Whereas virulent B. pertus-
sis induces a paroxysmal cough-like syndrome
when imbedded in agarose beads and admin-
istered intrabronchially to rats, a mutant that is
deficient in PTX secretion fails to do so, point-
ing to a central role of PTX for the induction of
cough. Whether this translates also to a similar
role of the toxin in human pertussis remains as
yet unknown.
More recently, a baboon model was developed
for the study of whooping cough [70] . This non-
human primate model is thought to more closely
mimic human pertussis, compared with all other
animal models. Unlike other primate models,
such as rhesus macaques, inoculation of olive
baboons with virulent B. pertussis resulted in
100% of the cases in clinical pertussis, including
leukocytosis and paroxysmal coughing. It will be
interesting to see the pathogenesis, especially the
cough, in baboons infected with PTX-deficient
B. pertussis mutants.
246 Future Microbiol. (2015) 10(2)
PTX inhibits host defense
One potential mechanism by which PTX par-
ticipates in the early events of the infectious pro-
cess is its ability to inhibit neutrophil influx into
the respiratory tract of B. pertussis-infected hosts
in the very early stages of infection, but not in
the later stages [71] . In addition, PTX may target
resident airway macrophages, as a depletion of
the airway macrophages by intranasal admin-
istration of liposome-encapsulated clodronate
prior to infection was shown to enhance B. per-
tussis colonization in mice in the early stages.
This enhancement by the clodronate treatment
was also seen when a PTX-deficient mutant
was used for infection and could be directly
linked to PTX-catalyzed ADP-ribosylation of
airway macrophage G proteins [72] . PTX also
alters the expression profile of resident airway
macrophages and inhibits the production of
certain chemokines in response to the infection
in mice. Especially the expression of keratino-
cyte-derived chemokines and LPS-stimulated
CXC chemokines are downregulated by infec-
tion with PTX-producing B. pertussis. Again,
this downregulation depends on the enzyme
activity of the toxin [73] . This, in turn, affects
the recruitment of neutrophils to the airways,
as these chemokines constitute the major
­neutrophil-attracting chemokines. The addition
of exogenous keratinocyte-derived chemokine
enhanced the early neutrophil recruitment dur-
ing B. pertussis infection. In addition to airway
macrophages, other cell types, including epithe-
lial cells in the respiratory tract, may also partici-
pate in the production of neutrophil-attracting
chemokines, and their expression may also be
downregulated by PTX.
The role of PTX in the inhibition of neu-
trophil recruitment to the airways may thus be
indirect via its action on macrophages and/or epi-
thelial cells, rather than via a direct action on the
neutrophils themselves. However, PTX was also
found to directly ADP-ribosylate the Gi proteins
associated with chemokine receptors on the neu-
trophils themselves, which can also result in the
inhibition of neutrophil migration [74] . However,
the role in the pathogenesis of pertussis of this
direct action on the neutrophils is not clear.
Surprisingly, depletion of neutrophils has no
impact on B. pertussis infection of naive mice.
A protective role of the neutrophils during
infection was only observed when the mice had
already been previously infected or had received
immune serum [75] . This may be due to the
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presence of opsonizing antibodies against B. per-
tussis, which might increase bacterial uptake by
phagocytosis and subsequent killing by the
neutrophils. However, even in the absence of
neutrophils, PTX still plays a role in infection,
thereby suggesting that other cells, such as the
airway macrophages may be major targets for
the toxin.
PTX modulates innate & adaptive immune
responses
Transcriptomic studies on a variety of human
cell types, including bronchial epithelial cell
lines, lung fibroblasts, endothelial cells, pulmo-
nary artery smooth muscle cells and immature
monocyte-derived dendritic cells, incubated
with PTX revealed that the toxin only induced
differential gene expression in the latter cells.
Essentially the expression of six genes, those
coding for IFN-γ, IL-2, XCL1, CD69, CSF2
and CXCL10, was significantly upregulated by
incubation with PTX [76] . As dendritic cells are
major antigen-presenting cells and represent the
cornerstone between innate and adaptive immu-
nity, these observation suggest that PTX may
have a major effect in the perturbation of the
host immune system during infection.
Although most of the PTX activities on
immune cells can be ascribed to its ADP-
ribosylation activity, the enzymatically inactive
B oligomer by itself may also induce profound
changes in those cells. It has been reported that
T cells treated with the B oligomer fail to initiate
signal transduction in response to macrophage
inflammatory protein-1ß or RANTES, with-
out impairing its binding to the cell surface via
the receptor CCR5 [77] . Instead, the effect of
the B oligomer on CCR5-mediated signaling is
reversed by protein kinase C inhibitors. The B
oligomer also causes desensitization of the related
chemokine receptor CXCR4, which is targeted
by the stromal cell-derived factor 1α. CXCR4-
dependent signaling induces T-cell migration,
and treatment with the PTX B oligomer blocks
stromal cell-derived factor 1α-induced chemo-
taxis [78] . Furthermore, unlike CCR5, CXCR4
surface expression is decreased upon treatment
with the B oligomer. These effects were shown to
be mediated by the B oligomer-induced a­ ctivation
of the TCR signaling network.
Enzymatically inactive PTX induces the mat-
uration of human monocyte-derived dendritic
cells, presumably via the activities of the B oli-
gomer [79] and protects them from spontaneous
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Investigating pertussis toxin & its impact on vaccination  Review
apoptosis. It induces the production of high lev-
els of IL-12p70, IL-1ß, IL-6, IL-23 and IL-10
by mature dendritic cells. This effect occurs
through PTX-stimulated TLR-4 signaling,
and, to a minor extend, perhaps TLR-2 signal-
ing as well. This, in turn drives the expansion
of Th1 and Th17 T cells. However, it has been
reported that some of the described activities of
PTX, such as the induction of proinflamma-
tory cytokines by human blood cells, may have
resulted from a potential contamination of the
PTX preparations with LPS [80] .
Both infection with B. pertussis and vaccina-
tion with wPV induce Th1 and Th17 responses.
In TLR-4-defective mice, wPV fail to elicit pro-
tective immunity, which is paralleled by reduced
antigen-specific IL-17 and IFN-γ production, as
compared with TLR-4 + mice [81] . In mice IL-17
also appears to be essential for the induction of
protective immunity by aPV, as in IL-1A-defective
mice, aPV fails to elicit protection. aPV-mediated
bacterial clearance depends on Th17 cell recruit-
ment to the lungs after challenge. Alum, present
as adjuvant in aPV, appears to promote IL-17A
production via an IL-1ß-dependent mechanism
[82] . Mucosal IL-17, in addition to IL-6 and IL-23,
could also be measured in high quantities in the
nasopharynx of B. pertussis-infected baboons [83]
and antigen-specific Th17, as well as Th1 cells
were still found at high levels up to 2 years after
infection. These observations suggest that Th17
and Th1 cells also contribute to long-lasting
immunity in primates after infection.
In addition to Th1 and Th17 T cells involved
in protective immunity against B. pertussis,
antibodies also play a major role, and PTX is
known for long to potentiate local and systemic
antibody production. Initially, PTX was known
to strongly enhance the production of IgE to
co-administered antigens [84] . It substantially
increases the frequency of IgE-bearing lympho-
cytes in spleen and blood when co-administered
with an antigen such as egg albumin. When co-
administered with tetanus toxoid or diphtheria
toxoid, PTX strongly increases IgG1 and IgE
titers to these antigens. This adjuvant activity
appears to depend on IL-4-producing T cell
priming by the toxin [85] .
Enzymatically inactive PTX also induces
high levels of IgA and IgG to antigens co-
administered by mucosal routes. A combination
of tetanus toxin fragment C with enzymatically
inactive PTX delivered nasally resulted in high
antibody titers against tetanus toxin, even after
www.futuremedicine.com 247
Review  Coutte & Locht
a single nasal administration [86] . This was not
seen when the antigen was combined with enzy-
matically active PTX. High levels of IgA were also
detected in the nasal washes after administration
of the antigen with inactive or with active toxin.
Interestingly, IgE was only produced when the
antigen was combined with active toxin, but not
when it was combined with enzymatically inac-
tive PTX. These observations indicate that the
adjuvant activity of the toxin relies on two dif-
ferent mechanisms, one depending on its ADP-
ribosyltransferase activity, such as for the induc-
tion of IgE, and one independent of this enzyme
activity.
Treatment by PTX also results in a strong
reduction in the frequency and in the immuno-
suppressive activity of CD4 +CD25+FoxP3 + regu-
latory T cells in a model of experimental autoim-
mune encephalitis [87] . This property may also
contribute to its adjuvant activity and was shown
to depend on the ADP-ribosyltransferase activity.
Strangely, the suppressive activity of the regula-
tory T cells was not inhibited by PTX in vitro, but
was clearly inhibited in vivo. The precise mecha-
nism and the specific molecular targets of this
activity remain to be investigated. Recently, the
ability of PTX to decrease the number of regu-
latory T cells was exploited in an experimental
model of immunotherapy against C6 glioma in
rats [88] .
Paradoxically, in the context of the whole bac-
teria, the toxin appears to be immunosuppres-
sive. PTX-producing B. pertussis strains induce
substantially lower titers of serum antibodies
to FHA or to heterologous antigens fused to
FHA upon intranasal infection than their iso-
genic strains that lack the toxin gene [89] . This
decrease in serum antibody titers was dependent
on the presence of S1, suggesting a role of the
ADP-ribosytransferase activity on the immuno-
suppressive properties of PTX. This immuno-
suppressive effect is not specifically targeted to
FHA, but was observed for various B. pertussis
antigens [90] . In particular, an immundominant
lipoprotein was strongly recognized by antibodies
from mice infected with PTX-deficient strains,
but hardly any antibody response to this antigen
was detected when the mice were infected with
toxin-producing strains. The addition of purified
PTX to the toxin-deficient mutant inoculum led
again to a reduction of anti-B. pertussis serum
antibodies, confirming the specific role of PTX
in the immunosuppressive effect. Whether this
immunosuppressive effect of PTX is also reflected
248 Future Microbiol. (2015) 10(2)
in the T cell responses or in the mucosal immune
responses has not been investigated yet.
Pathogen adaptation & conclusion
With the exception of Poland, all European coun-
tries have now replaced wPV by aPV (for a recent
review, see [91]), and vaccination coverage is very
high in most countries. Nevertheless, many coun-
tries in the Western world have witnessed recently
a dramatic increase in the incidence of whooping
cough, which makes it today the most prevalent
vaccine-preventable disease in industrialized
countries. Reasons for this increase may be mul-
tiple and include fast waning of vaccine-induced
immunity and pathogen adaptation [92,93] . The
most recent B. pertussis strains show genetic
changes, mainly single-nucleotide polymorphisms
(SNPs) located in regions involved in transcrip-
tion and translation of virulence genes [94,95] . SNP
typing permits the classification of the B. pertussis
strains into six major groups, named clusters I to
VI. Cluster I has a worldwide distribution and
originated probably from ancestor strains before
the aPV were introduced. Strains from the others
clusters, presenting changes in genes encoding
the acellular vaccine antigens PTX, Prn, Fim2
and Fim3 appear to reflect the genetic evolution
of more recent clinical isolates [96] .
The SNPs located in the PTX promoter
region can also be used for the classification of
the emergent strains in clusters named ptxP1,
ptxP2, ptxP3 and ptxP4 [97] . These strains also
showed SNPs changes in the other virulence
genes. Strains with the ptxP1 and ptxP3 alleles
are largely overrepresented among typed tested
clinical isolates, suggesting that they are perhaps
more virulent in humans than the others [97–99] .
Whereas ptxP1 strains were frequently isolated
before aPV immunization, strains carrying the
ptxP3 allele were isolated more recently. These
strains were shown to produce higher amounts
of PTX and to be more virulent in an intranasal
mouse infection model than the other strains
[99,100] . The increase in levels of PTX production
may thus potentially help the micro-organism to
overcome aPV-induced immunity. Very recently,
the number of clinical isolates that have lost the
gene coding for Prn, another major antigen in
aPV, also has been increasing dramatically [101] .
The loss of the prn gene in current B. pertussis
strains under aPV pressure strongly suggests that
Prn is a major protective antigen in aPV, and,
in apparent contrast, that B. pertussis can be a
successful pathogen, even in the absence of Prn.
future science group

In fact, Prn-negative strains are not less virulent
in humans than Prn-producing strains [102] . A
clinical PTX-deficient B. pertussis isolate has also
been reported. However, this strain was signifi-
cantly less virulent in mouse models and caused
a milder pertussis-like disease, compared with
PTX-producing strains [103] .
These observations illustrate that antigenic
escape by the deletion of nonessential genes cod-
ing for antigens present in the current aPV is a
likely pathogen adaptation strategy for B. per-
tussis. Antigenic escape is more likely a patho-
gen response to vaccines containing only a few
components, such as the aPV, than to vaccines
that contain thousands of different antigens, such
as wPV. However, it is unlikely that aPV-using
countries will make the switch back to the first-
generation wPV.
An alternative may be the recently developed
live attenuated nasal vaccine, based on the genetic
detoxification or removal of three B. pertussis tox-
ins, the dermonecrotic toxin, the tracheal cyto-
toxin and PTX [104] . The target population of this
vaccine could be the neonates, and murine studies
have shown that a single nasal administration of
this vaccine to infant mice induces long-lasting
immunity against experimental B. pertussis chal-
lenge infection, for up to at least one year after
immunization [105] . In addition to provide full pro-
tection against pertussis challenge in mice, this
vaccine also cross-protects against challenge with
Bordetella parapertussis and Bordetella bronchisep-
tica [106] . In addition, it has interesting bystander
effects, as it also protects against influenza virus-
induced pneumonia [107] respiratory syncytial virus
disease [108] and allergic asthma [109] . This live
nasal vaccine has now undergone a human Phase I
clinical trial and was found to induce immune
responses in all colonized individuals without
causing any vaccine-related adverse event [110] .
Although this appears as a promising approach for
the control of pertussis and perhaps for protection
against other respiratory illnesses, many hurdles
have to be overcome before it can be made widely
­available for neonatal vaccination [111] .
Future perspective
Although the precise role of PTX in the patho-
genesis of human whooping cough is still not
clear, many studies in mouse models point to
important roles in colonization of the respiratory
tract, in pathogenesis, as well as in immune dys-
function and consequently in immune escape of
the micro-organism during infection. The recent
future science group
Investigating pertussis toxin & its impact on vaccination  Review
global transcriptomic approaches have identified
several hundreds of genes that are up- or downreg-
ulated specifically upon infection of mice by PTX-
producing B. pertussis in contrast to PTX-deficient
strains [68] . This provides a wealth of information
that will be helpful to decipher at the molecular
and cellular levels the key pathogenic mechanisms
induced by PTX during the infectious process.
One of the most recently studied novel host pro-
teins involved in the PTX-dependent pathology
induced by B. pertussis infection in mice is the epi-
thelial anion transporter protein pendrin, indeed
first identified by transcriptomic analyses. This
protein was recently shown to contribute to the
inflammatory lung pathology in infected mice
[112] . It is likely that similar approaches will con-
tribute in the near future to a more comprehen-
sive view on the pathological involvement of PTX
­during B. pertussis infection in mice.
It will also be interesting to see whether PTX-
producing B. pertussis triggers specific epigenetic
reprograming, compared with PTX-deficient
strains. It is well recognized that epigenetics,
essentially mediated by inheritable DNA meth-
ylation and histone modifications, can strongly
influence gene expression through changes in
chromatin structure. Epigenetic modifications of
DNA and histones are known to regulate T cell
differentiation and the expression of effector mol-
ecules and cell surface markers on immune cells
[113] . Certain bacterial infections have been docu-
mented to induce epigenetic changes via histone
acetylation/deacetylation and phosphorylation/
dephosphorylation or via aberrant DNA meth-
ylation [114] . Although not yet studied in the con-
text of B. pertussis infection, it would not be too
surprising if some of the induced gene expression
profiles seen upon B. pertussis infection are accom-
panied by changes in chromatin structure, and,
if so, some of those may very well depend on the
action of PTX. This would open a new area of
investigation on pertussis pathogenesis.
Although mouse models have been useful to
decipher molecular and cellular events triggered
by B. pertussis infection, they do not reproduce
the full spectrum of human pertussis, including
the cough syndrome, and are therefore of limited
use to study this disease. The recent development
of a baboon model [70] provides a more relevant
model and would be expected to shed new light
on the function of PTX in the pathogenesis of
whooping cough in an animal that is more closely
related to humans. This model has already shown
that aPV containing PTX are able to protect
www.futuremedicine.com 249
Review  Coutte & Locht

EXECUTIVE SUMMARY
Pertussis vaccines & the discovery of pertussis toxin as a protective antigen
●● The history of the pertussis vaccines development: the replacement of the whole-cell vaccines (wPV) by the less
reactogenic acellular vaccines (aPV).
●● The development and composition of the aPV.
●● The pertussis toxin (PTX) is described as a monocomponent PTX vaccine.
The structure of PTX
●● Gene composition and expression of the PTX in the Bordetellae.
●● The description of the ADP-ribosylating activities of the PTX.
●● The PTX is a pyramid-shaped molecule belonging to the A-B family of toxins.
Biogenesis of PTX
●● The genes coding for the PTX biogenesis and secretion belong to the same operon and are regulated by the BvgA/S
virulence regulation system of Bordetella pertussis.
●● The PTX is secreted by a type IV secretion system, the Ptl machinery, through the bacterial envelope.
Toxin binding, trafficking & enzymatic activities on the host target cells
●● The binding of the PTX to its glyco-conjugates receptors is mediated by different domains present on the S2 and S3
subunits.
●● The PTX entry in the targeted cell involved a receptor-mediated endocytosis followed by retrograde transport in the
Golgi apparatus and the endoplasmic reticulum (ER).
Roles of PTX during infection
●● The PTX intoxicates the targeted cell by ADP-ribosylation of Giα proteins.
●● The mouse models of infection reveal the role of PTX at the early stages of infection.
●● Role of the PTX in pulmonary inflammation during infection.
●● The PTX is involved in the leukocytosis and coughing symptoms but the recently described baboon model may be a
best model to decipher precisely all the PTX related symptoms during the disease.
PTX inhibits host defense
●● The PTX inhibits directly or indirectly the influx and functionalities of the airway immune cells.
PTX modulates innate & adaptive immune responses
●● The PTX modifies the transcriptomic pattern of intoxicated cells.
●● The protective immunity against B. pertussis seems to be mediated by Th1 and Th17 responses.
●● The PTX owns some strong adjuvant activities.
●● Immunosuppressive activities of the PTX.
Pathogen adaptation & conclusion
●● Some recent B. pertussis strains isolates show some bacterial adaptation as changes in genes encoding for some of the
acellular vaccine antigenes such as pertactin and PTX.
●● Some of these emergent strains show an increase in PTX production and are more virulent in an intranasal mouse
model of infection.
●● Development of a live attenuated Bordetella pertussis strain as a new nasal vaccine.

250 Future Microbiol. (2015) 10(2) future science group


EXECUTIVE SUMMARY (CONT.)
Future perspectives
●● The description of the transcriptomic approaches used to identify the role of PTX during infection.
●● Some hypothetical epigenetic modifications could occur during B. pertussis infection.
●● The baboon model would be a better model to study the clinical pertussis disease.
●● Shift from chemically detoxified to a genetically detoxified PTX in aPV.
these primates against pertussis disease, includ-
ing cough, but are not able to protect against
infection by B. pertussis and transmission of the
organism [115] , illustrating one of the important
shortcomings of the current aPV.
Meanwhile, PTX is the main component of
all currently available vaccines, and the presence
of antibodies to this toxin has been correlated
with protection against the most severe forms of
the disease in children [116] . It is thus likely that
PTX will continue to be included as an essential
component in pertussis vaccines. However, the
detrimental effects of formaldehyde/glutaral-
dehyde treatments of the toxin on its antigenic
structure, and the ability to detoxify PTX by
genetic modifications of its structural genes may
perhaps motivate vaccine manufacturers in the
future to shift from a chemically detoxified aPV
to a genetically detoxified aPV. This has already
shown safety, superior immunogenicity over
chemically detoxified PTX and the induction of
References
1 Paddock CD, Sanden GN, Cherry JD et al.
Pathology and pathogenesis of fatal Bordetella
pertussis infection in infants. Clin. Infect.
Dis. 47(3), 328–338 (2008).
2 Von Konig CH, Halperin S, Riffelmann M,
Guiso N. Pertussis of adults and infants.
Lancet Infect. Dis. 2(12), 744–750 (2002).
3 Storsaeter J, Wolter J, Locht C. Pertussis
vaccines. In: Bordetella Molecular
Microbiology. Locht C (Ed.). Horizon
Bioscience, Norfolk, UK, 245–288 (2007).
4 Sheridan Sl, Ware RS, Grimwood K, Lambert
SB. Number and order of whole cell pertussis
vaccines in infancy and disease protection.
JAMA 308(5), 454–456 (2012).
5 He Q, Mertsola J. Factors contributing to
pertussis resurgence. Future Microbiol. 3(3),
329–339 (2008).
6 Bordet J, Gengou O. Le microbe de la
coqueluche. Ann. Inst. Pasteur. 20, 731–741
(1906).
future science group
Investigating pertussis toxin & its impact on vaccination  Review
long-lasting immunity in man more than 20 years
ago [117] . It may thus perhaps be time to go back to
the future. However, even with a shift to geneti-
cally detoxified PTX, aPV may still not be able to
protect against B. pertussis infection. To achieve
this goal, an entirely different type of pertussis
vaccine is likely to be needed, and perhaps the
development of a mucosally applied live attenu-
ated vaccine may represent the long term solution
to the global pertussis problem [118] .
Financial & competing interests disclosure
The authors have no relevant affiliations or financial
involvement with any organization or entity with a finan-
cial interest in or financial conflict with the subject matter
or materials discussed in the manuscript. This includes
employment, consultancies, honoraria, stock ownership or
options, expert testimony, grants or patents received or pend-
ing, or royalties.
No writing assistance was utilized in the production of
this manuscript.
7 Sato Y, Kimura M, Fukumi H. 12 Locht C, Keith JM. Pertussis toxin gene:
Development of a pertussis component nucleotide sequence and genetic organization.
vaccine in Japan. Lancet 8369(1), 122–126 Science 232, 1258–1264 (1986).
(1984). 13 Locht C, Lobet Y, Feron C, Cieplak W, Keith
8 Sato Y, Arai H, Suzuki K. Leukocytosis- JM. The role of cysteine 41 in the enzymatic
promoting factor of Bordetella pertussis. II: activities of the pertussis toxin S1 subunit as
biological properties. Infect. Immun. 7(6), investigated by site-directed mutagenesis.
992–999 (1973). J. Biol. Chem. 265(8), 4552–4559 (1990).
9 Parfentjev IA, Goodline MA. Histamine 14 Cortina G, Barbieri JT. Role of tryptophan
shock in mice sensitized with Hemophilus 26 in the NAD glycohydrolase reaction of the
pertussis vaccine. J. Pharmacol. Exp. S-1 subunit of pertussis toxin. J. Biol.
Ther. 92(4), 411–413 (1948). Chem. 264(29), 17322–17328 (1989).
10 Munoz JJ, Arai H, Bergman RK, 15 Locht C, Capiau C, Feron C. Identification of
Sadowski PL. Biological activities of amino acid residues essential for the
crystalline pertussigen from Bordetella enzymatic activities of pertussis toxin. Proc.
pertussis. Infect. Immun. 33(3), 820–826 Natl Acad. Sci. USA 86(9), 3075–3079
(1981). (1989).
11 Ibsen PH. The effect of formaldehyde, 16 Burnette WN, Cieplak W, Mar VL, Kaljot
hydrogen peroxide and genetic detoxification KT, Sato H, Keith JM. Pertussis toxin S1
of pertussis toxin on epitope recognition by mutant with reduced enzyme activity and a
murine monoclonal antibodies. conserved protective epitope.
Vaccine 14(5), 359–368 (1996). Science 242(4875), 72–74 (1988).
www.futuremedicine.com 251
Review  Coutte & Locht
17 Antoine R, Locht C. The NAD-
glycohydrolase activity of the pertussis toxin
S1 subunit. Involvement of the catalytic
His-35 residue. J. Biol. Chem. 269(9),
6450–6457 (1994).
18 Antoine R, Tallett A, Van Heyningen S,
Locht C. Evidence for a catalytic role of
glutamic acid 129 in the NAD-glycohydrolase
activity of the pertussis toxin S1 subunit.
J. Biol. Chem. 268(32), 24149–24155 (1993).
19 Pizza M, Covacci A, Bartoloni A et al.
Mutants of pertussis toxin suitable for vaccine
development. Science 246(4929), 497–500
(1989).
20 Nencioni L, Pizza M, Bugnoli M et al.
Characterization of genetically inactivated
pertussis toxin mutants: candidates for a new
vaccine against whooping cough. Infect.
Immun. 58(5), 1308–1315 (1990).
21 Zhang JM, Cowell JL, Steven AC, Manclark
CR. Purification of serotype 2 fimbriae of
Bordetella pertussis and their identification as a
mouse protective antigen. Dev. Biol.
Stand. 61, 173–185 (1985).
22 Shahin RD, Brennan MJ, Li ZM, Meade BD,
Manclark CR. Characterization of the
protective capacity and immunogenicity of the
69-kD outer membrane protein of Bordetella
pertussis. J. Exp. Med. 171(1), 63–73 (1990).
23 Olin P, Rasmussen F, Gustafsson L,
Hallander HO, Heijbel H. Randomised
controlled trial of two-component, three-
component, and five-component acellular
pertussis vaccines compared with whole-cell
pertussis vaccine. Lancet 350(9091),
1569–1577 (1997).
24 Trollfors B, Taranger J, Lagergard T et al. A
placebo-controlled trial of a pertussis-toxoid
vaccine. N. Engl. J. Med. 333(16), 1045–1050
(1995).
25 Hviid A, Stellfeld M, Andersen PH,
Wohlfahrt J, Melbye M. Impact of routine
vaccination with a pertussis toxoid vaccine in
Denmark. Vaccine 22(27–28), 3530–3534
(2004).
26 Robbins JB, Schneerson R, Kubler-Kielb J
et al. Toward a new vaccine for pertussis. Proc.
Natl Acad. Sci. USA 111(9), 3213–3236
(2014).
27 Marchitto KS, Smith SG, Locht C, Keith JM.
Nucleotide sequence homology to pertussis
toxin gene in Bordetella bronchiseptica and
Bordetella parapertussis. Infect. Immun. 55(3),
497–501 (1987).
28 Arico B, Rappuoli R. Bordetella parapertussis
and Bordetella bronchiseptica contain
transcriptionally silent pertussis toxin genes.
J. Bacteriol. 169(6), 2847–2853 (1987).
252 Future Microbiol. (2015) 10(2)
29 Stefanelli P, Mastrantonio P, Hausman SZ,
Giuliano M, Burns DL. Molecular
characterization of two Bordetella
bronchiseptica strains isolated from children
with coughs. J. Clin. Microbiol. 35(6),
1550–1555 (1997).
30 Katada T, Ui M. ADP ribosylation of the
specific membrane protein of C6 cells by
islet-activating protein associated with
modification of adenylate cyclase activity. J.
Biol. Chem. 257(12), 7210–7216 (1982).
31 Bokoch GM, Katada T, Northup JK, Hewlett
EL, Gilman AG. Identification of the
predominant substrate for ADP-ribosylation
by islet activating protein. J. Biol.
Chem. 258(4), 2072–2075 (1983).
32 Locht C, Coutte L, Mielcarek N. The ins and
outs of pertussis toxin. FEBS J. 278(23),
4668–4682 (2011).
33 Tamura M, Nogimori K, Murai S et al.
Subunit structure of islet-activating protein,
pertussis toxin, in conformity with the A-B
model. Biochemistry 21(22), 5516–5522
(1982).
34 Stein PE, Boodhoo A, Armstrong GD,
Cockle SA, Klein MH, Read RJ. The crystal
structure of pertussis toxin. Structure 2(1),
45–57 (1994).
35 Nicosia A, Perugini M, Franzini C et al.
Cloning and sequencing of the pertussis toxin
genes: operon structure and gene duplication.
Proc. Natl Acad. Sci. USA 83(13), 4631–4635
(1986).
36 Raze D, Veithen A, Sato H, Antoine R,
Menozzi FD, Locht C. Genetic exchange of
the S2 and S3 subunits in pertussis toxin.
Mol. Microbiol. 60(5), 1241–1250 (2006).
37 Weiss AA, Melton AR, Burns DL. Molecular
characterization of an operon required for
pertussis toxin secretion. Proc. Natl Acad. Sci.
USA 90, 2970–2974 (1993).
38 Roy CR, Miller JF, Falkow S. The bvgA gene
of Bordetella pertussis encodes a transcriptional
activator required for coordinate regulation of
several virulence genes. J. Bacteriol. 171(11),
6338–6344 (1989).
39 Farizo KM, Fiddner S, Cheung AM, Burns
DL. Membrane localization of the S1 Subunit
of pertussis toxin in Bordetella pertussis and
implications for pertussis toxin secretion.
Infect. Immun. 70(3), 1193–1201 (2002).
40 Antoine R, Locht C. Roles of the disulfide
bond and the carboxy-terminal region of the
S1 subunit in the assembly and biosynthesis of
pertussis toxin. Infect. Immun. 58(6),
1518–1526 (1990).
41 Farizo KM, Huang T, Burns DL. Importance
of holotoxin assembly in Ptl-mediated
secretion of pertussis toxin from Bordetella
pertussis. Infect. Immun. 68(7), 4049–4054
(2000).
42 Farizo KM, Cafarella TG, Burns DL.
Evidence for a ninth gene, ptlI, in the locus
encoding the pertussis toxin secretion system
of Bordetella pertussis and formation of a
PtlI-PtlF complex. J. Biol. Chem. 271(49),
31643–31649 (1996).
43 Craig-Mylius KA, Weiss AA. Mutants in the
ptlA-H genes of Bordetella pertussis are
deficient for pertussis toxin secretion. FEMS
Microbiol. Lett. 179(2), 479–484 (1999).
44 Cook DM, Farizo KM, Burns DL.
Identification and characterization of PtlC: an
essential component of the pertussis toxin
secretion system. Infect. Immun. 67(2),
754–759 (1999).
45 Kotob SI, Burns DL. Essential role of the
consensus nucleotide-binding site of PtlH in
secretion of pertussis toxin from Bordetella
pertussis. J. Bacteriol. 179(23), 7577–7580
(1997).
46 Craig-Mylius KA, Stenson TH, Weiss AA.
Mutations in the S1 subunit of pertussis toxin
that affect secretion. Infect. Immun. 68(3),
1276–1281 (2000).
47 Armstrong GD, Howard LA, Peppler MS.
Use of glycosyltransferases to restore pertussis
toxin receptor activity to
asialoagalactofetuin. J. Biol. Chem. 263(18),
8677–8684 (1988).
48 Brennan MJ, David JL, Kenimer JG,
Manclark CR. Lectin-like binding of pertussis
toxin to a 165-kilodalton Chinese hamster
ovary cell glycoprotein. J. Biol.
Chem. 263(10), 4895–4899 (1988).
49 Bogdan JA, Yuan W, Long-Rowe KO,
Sarwar J, Brucker EA, Blake MS.
Identification of peptides that mimic the
pertussis toxin binding site on bovine fetuin.
Appl. Env. Microbiol. 69(10), 6272–6279
(2003).
50 Stein PE, Boodhoo A, Armstrong GD et al.
Structure of a pertussis toxin-sugar complex
as a model for receptor binding. Nat. Struct.
Biol. 1(9), 591–596 (1994).
51 Lobet Y, Feron C, Dequesne G, Simoen E,
Hauser P, Locht C. Site-specific alterations in
the B oligomer that affect receptor-binding
activities and mitogenicity of pertussis toxin.
J. Exp. Med. 177(1), 79–87 (1993).
52 Millen SH, Lewallen DM, Herr AB, Iyer SS,
Weiss AA. Identification and characterization
of the carbohydrate ligands recognized by
pertussis toxin via a glycan microarray and
surface plasmon resonance.
Biochemistry 49(28), 5954–5967 (2010).
future science group

53 Xu Y, Barbieri JT. Pertussis toxin-mediated
ADP-ribosylation of target proteins in Chinese
hamster ovary cells involves a vesicle
trafficking mechanism. Infect. Immun. 63(3),
825–832 (1995).
54 Xu Y, Barbieri JT. Pertussis toxin-catalyzed
ADP-ribosylation of Gi-2 and Gi-3 in CHO
cells is modulated by inhibitors of intracellular
trafficking. Infect. Immun. 64(2), 593–599
(1996).
55 Plaut RD, Carbonetti NH. Retrograde
transport of pertussis toxin in the mammalian
cell. Cell. Microbiol. 10(5), 1130–1139 (2008).
56 Hausman SS, Burns DL. Interaction of
pertussis toxin with cells and model
membranes. J. Biol. Chem. 267(19),
13735–13739 (1992).
57 Hazes B, Read RJ. Accumulating evidence
suggests that several AB-toxins subvert the
endoplasmic reticulum-associated protein
degradation pathway to enter target cells.
Biochemistry 36(37), 11051–11054 (1997).
58 Worthington ZEV, Carbonetti NH. Evading
the proteasome: absence of lysine residues
contributes to pertussis toxin activity by
evasion of proteasome degradation. Infect.
Immun. 75(6), 2946–2953 (2007).
59 Locht C, Antoine R. A proposed mechanism
of ADP-ribosylation catalyzed by the pertussis
toxin S1 subunit. Biochimie 77(5), 333–340
(1995).
60 Pittman M. The concept of pertussis as a
toxin-mediated disease. Pediatr. Infect. Dis.
J. 3(5), 467–486 (1984).
61 Loosmore S, Zealey G, Cockle S et al.
Characterization of pertussis toxin analogs
containing mutations in B-oligomer subunits.
Infect. Immun. 61(6), 2316–2324 (1993).
62 Diehl SA, Mcelvany B, Noubade R et al. G
proteins Gai1/3 are critical targets for
Bordetella pertussis toxin-induced vasoactive
amine sensitization. Infect. Immun. 82(2),
773–782 (2014).
63 Carbonetti NH, Artamonova GV, Mays RM,
Worthington ZE. Pertussis toxin plays an
early role in respiratory tract colonization by
Bordetella pertussis. Infect. Immun. 71(11),
6358–6366 (2003).
64 Pittman M. Pertussis toxin: the cause of the
harmful effects and prolonged immunity of
whooping cough: a hypothesis. Rev. Infect.
Dis. 1(3), 401–412 (1979).
65 Alonso S, Pethe K, Mielcarek N, Raze D,
Locht C. Role of ADP-ribosyltransferase
activity of pertussis toxin in toxin-adhesin
redundancy with filamentous hemagglutinin
during Bordetella pertussis infection. Infect.
Immun. 69(10), 6038–6043 (2001).
future science group
Investigating pertussis toxin & its impact on vaccination  Review
66 Relman D, Tuomanen E, Falkow S,
Golenbock DT, Saukkonen K, Wright SD.
Recognition of a bacterial adhesin by an
integrin: macrophage CR3 (aMß2, CD11b
CD18) binds filamentous hemagglutinin of
Bordetella pertussis. Cell 61(7), 1375–1382
(1990).
67 Khelef N, Bachelet CM, Vargaftig BB, Guiso
N. Characterization of murine lung
inflammation after infection with parental
Bordetella pertussis and mutants deficient in
adhesins or toxins. Infect. Immun. 62(7),
2893–2900 (1994).
68 Connelly CE, Sun Y, Carbonetti NH.
Pertussis toxin exacerbates and prolongs
airway inflammatory responses during
Bordetella pertussis infection. Infect
Immun. 80(12), 4317–4332 (2012).
69 Parton R, Hall E, Wardlaw AC. Responses to
Bordetella pertussis mutant strains and to
vaccination in the coughing rat model of
pertussis. J. Med. Microbiol. 40(5), 307–312
(1994).
70 Warfel JM, Beren J, Kelly VK, Lee G, Merkel
TJ. Nonhuman primate model of pertussis.
Infect. Immun. 80(4), 1530–1536 (2012).
71 Carbonetti NH, Artamonova GV, Andreasen
C, Bushar N. Pertussis toxin and adenylate
cyclase toxin provide a one-two punch for
establishment of Bordetella pertussis infection
of the respiratory tract. Infect. Immun. 73(5),
2698–2703 (2005).
72 Carbonetti NH, Artamonova GV, Van
Rooijen N, Ayala VI. Pertussis toxin targets
airway macrophages to promote Bordetella
pertussis infection of the respiratory tract.
Infect. Immun. 75(4), 1713–1720 (2007).
73 Andreasen C, Carbonetti NH. Pertussis toxin
inhibits early chemokine production to delay
neutrophil recruitment in response to
Bordetella pertussis respiratory tract infection
in mice. Infect. Immun. 76(11), 5139–5148
(2008).
74 Kirimanjeswara GS, Agosto LM, Kennett MJ,
Bjornstad ON, Harvill ET. Pertussis toxin
inhibits neutrophil recruitment to delay
antibody-mediated clearance of Bordetella
pertussis. J. Clin. Invest. 115(12), 3594–3601
(2005).
75 Andreasen C, Carbonetti NH. Role of
neutrophils in response to Bordetella pertussis
infection in mice. Infect. Immun. 77(3),
1182–1188 (2009).
76 Vaessen SF, Verkoeijen S, Vandebriel RJ et al.
Identification of biomarkers to detect residual
pertussis toxin using microarray analysis of
dendritic cells. Vaccine 31(45), 5223–5231
(2013)
www.futuremedicine.com
77 Alfano M, Schmidtmayerova H, Amella CA,
Pushkarsky T, Bukrinsky M. The B-oligomer
of pertussis toxin deactivates CC chemokine
receptor 5 and blocks entry of M-tropic
HIV-1 strains. J. Exp. Med. 190(5), 597–606
(1999).
78 Schneider OD, Weiss AA, Miller WE.
Pertussis toxin signals through the TCR to
initiate cross-desensitization of the chemokine
receptor CXCR4. J. Immunol. 182(9),
5730–5739 (2009).
79 Nasso M, Fedele G, Spensieri F et al.
Genetically detoxified pertussis toxin
Induces Th1/Th17 immune response
through MAPKs and IL-10-dependent
mechanisms. J. Immunol. 183(3), 1892–1899
(2009).
80 Bache C, Spreitzer I, Becker B et al.
Bordetella pertussis toxin does not induce the
release of pro-inflammatory cytokines in
human whole blood. Med. Microbiol.
Immunol. 201(3), 327–335 (2012).
81 Higgins SC, Jarnicki AG, Lavelle EC, Mills
KH. TLR4 mediates vaccine-induced
protective cellular immunity to Bordetella
pertussis: role of IL-17-producing T cells.
J. Immunol. 177(11), 7980–7989 (2006).
82 Ross PJ, Sutton CE, Higgins S et al. Relative
contribution of Th1 and Th17 cells in
adaptive immunity to Bordetella pertussis:
towards the rational design of an improved
acellular pertussis vaccine. PLoS Pathog. 9(4),
e1003264 (2013).
83 Warfel JM, Merkel TJ. Bordetella pertussis
infection induces a mucosal IL-17 response
and long-lived Th17 and Th1 immune
memory cells in nonhuman primates.
Mucosal Immunol. 6(4), 787–796 (2013).
84 Munoz JJ, Peacock MG. Action of
pertussigen (pertussis toxin) on serum IgE
and on Fc epsilon receptors on lymphocytes.
Cell. Immunol. 127(2), 327–336 (1990).
85 Samore MH, Siber GR. Pertussis toxin
enhanced IgG1 and IgE responses to primary
tetanus immunization are mediated by
interleukin-4 and persist during secondary
responses to tetanus alone. Vaccine 14(4),
290–297 (1996).
86 Roberts M, Bacon A, Rappuoli R et al. A
mutant pertussis toxin molecule that lacks
ADP-ribosyltransferase activity, PT-9K/129G,
is an effective mucosal adjuvant for
intranasally delivered proteins. Infect.
Immun. 63(6), 2100–2108 (1995).
87 Chen X, Oppenheim JJ, Winkler-Pickett RT,
Ortaldo JR, Howard OM. Glucocorticoid
amplifies IL-2-dependent expansion of
functional FoxP3(+)CD4(+)CD25(+)
T regulatory cells in vivo and enhances their
253
Review  Coutte & Locht
capacity to suppress EAE. Eur. J.
Immunol. 36(8), 2139–2149 (2006).
88 Orozco-Morales M, Sanchez-Garcia FJ,
Guevara-Salazar P et al. Adjuvant
immunotherapy of C6 glioma in rats with
pertussis toxin. J. Cancer Res. Clin. Oncol.
138(1), 23–33 (2012).
89 Mielcarek N, Riveau G, Remoue F, Antoine
R, Capron A, Locht C. Homologous and
heterologous protection after single
intranasal administration of live attenuated
recombinant Bordetella pertussis. Nat.
Biotechnol. 16(5), 454–457 (1998).
90 Carbonetti NH, Artamonova GV, Andreasen
C, Dudley E, Mays RM, Worthington ZE.
Suppression of serum antibody responses by
pertussis toxin after respiratory tract
colonization by Bordetella pertussis and
identification of an immunodominant
lipoprotein. Infect. Immun. 72(6), 3350–3358
(2004).
91 Locht C, Mielcarek N. New pertussis
vaccination approaches: en route to protect
newborns? FEMS Immunol. Med.
Microbiol. 66(2), 121–133 (2012).
92 Mooi Fr, Na Vdm, De Melker He. Pertussis
resurgence: waning immunity and pathogen
adaptation: two sides of the same coin.
Epidemiol Infect 142(4), 685–694 (2014).
93 Schmidtke AJ, Boney KO, Martin SW, Skoff
TH, Tondella ML, Tatti KM. Population
diversity among Bordetella pertussis isolates,
United States, 1935–2009. Emerg. Infect.
Dis. 18(8), 1248–1255 (2012).
94 Van Loo IH, Mooi FR. Changes in the Dutch
Bordetella pertussis population in the first 20
years after the introduction of whole-cell
vaccines. Microbiology 148(Pt 7), 2011–2018
(2002).
95 Octavia S, Sintchenko V, Gilbert GL et al.
Newly emerging clones of Bordetella pertussis
carrying prn2 and ptxP3 alleles implicated in
Australian pertussis epidemic in 2008–2010.
J. Infect. Dis. 205(8), 1220–1224 (2012).
96 Octavia S, Maharjan RP, Sintchenko V et al.
Insight into evolution of Bordetella pertussis
from comparative genomic analysis: evidence
of vaccine-driven selection. Mol. Biol.
Evol. 28(1), 707–715 (2011).
97 Lam C, Octavia S, Bahrame Z, Sintchenko V,
Gilbert GL, Lan R. Selection and emergence
of pertussis toxin promoter ptxP3 allele in the
254 Future Microbiol. (2015) 10(2)
evolution of Bordetella pertussis. Infect. Genet.
Evol. 12(2), 492–495 (2012).
98 Bart MJ, Van Gent M, Van Der Heide HG
et al. Comparative genomics of prevaccination
and modern Bordetella pertussis strains. BMC
Genomics 11, 627 (2010).
99 Mooi Fr, Van Loo Ih, Van Gent M et al.
Bordetella pertussis strains with increased toxin
production associated with pertussis
resurgence. Emerg. Infect. Dis. 15(8),
1206–1213 (2009).
100 King AJ, Van Der Lee S, Mohangoo A, Van
Gent M, Van Der Ark A, Van De Waterbeemd
B. Genome-wide gene expression analysis of
Bordetella pertussis isolates associated with a
resurgence in pertussis: elucidation of factors
involved in the increased fitness of epidemic
strains. PLoS ONE 8(6), e66150 (2013).
101 Lam C, Octavia S, Ricafort L et al. Rapid
increase in pertactin-deficient Bordetella
pertussis isolates, Australia. Emerg. Infect.
Dis. 20(4), 626–633 (2014).
102 Bodilis H, Guiso N. Virulence of pertactin-
negative Bordetella pertussis isolates from
infants, France. Emerg. Inefct. Dis. 19(3),
471–474 (2013).
103 Bouchez V, Brun D, Cantinelli T, Dore G,
Njamkepo E, Guiso N. First report and
detailed characterization of B. pertussis isolates
not expressing pertussis toxin or pertactin.
Vaccine 27(43), 6034–6041 (2009).
104 Mielcarek N, Debrie AS, Raze D et al. Live
attenuated B. pertussis as a single-dose nasal
vaccine against whooping cough. PLoS
Pathog. 2(7), e65 (2006).
105 Feunou PF, Kammoun H, Debrie AS,
Mielcarek N, Locht C. Long-term immunity
against pertussis induced by a single nasal
administration of live attenuated B. pertussis
BPZE1. Vaccine 28(43), 7047–7053 (2010)
106 Kammoun H, Feunou PF, Foligne B et al.
Dual mechanism of protection by live
attenuated Bordetella pertussis BPZE1 against
Bordetella bronchiseptica in mice.
Vaccine 30(40), 5864–5870 (2012).
107 Li R, Lim A, Phoon MC et al. Attenuated
Bordetella pertussis protects against highly
pathogenic influenza A viruses by dampening
the cytokine storm. J. Virol. 84(14),
7105–7113 (2010).
108 Schnoeller C, Roux X, Sawant D et al.
Attenuated Bordetella pertussis vaccine
protects against respiratory syncytial virus
disease via an IL-17-dependent mechanism.
Am. J. Respir. Crit. Care Med. 189(2),
194–202 (2014).
109 Kavanagh H, Noone C, Cahill E, English K,
Locht C, Mahon BP. Attenuated Bordetella
pertussis vaccine strain BPZE1 modulates
allergen-induced immunity and prevents
allergic pulmonary pathology in a murine
model. Clin. Exp. Allergy 40(6), 933–941
(2010).
110 Thorstensson R, Trollfors B, Al-Tawil N et al.
A Phase I clinical study of a live attenuated
Bordetella pertussis vaccine – BPZE1: a single
centre, double-blind, placebo-controlled,
dose-escalating study of BPZE1 given
intranasally to healthy adult male volunteers.
PLoS ONE 9(1), e83449 (2014).
111 Meade BD, Plotkin SA, Locht C. Possible
options for new pertussis vaccines. J. Infect.
Dis. 209(Suppl. 1), S24–S27 (2014).
112 Scanlon KM, Gau Y, Zhu J et al. The
epithelial anion transporter pendrin
contributes to inflammatory lung pathology
in mouse models of Bordetella pertussis
infection. Infect. Immun. 82 (10) , 4212–4221
(2014).
113 Youngblood B, Hale JS, Ahmed R. T-cell
memory differentiation: insights from
transcriptional signatures and epigenetics.
Immunology 139(3), 277–284 (2013).
114 Bierne H, Hamon M, Cossart P. Epigenetics
and bacterial infections. Cold Spring Harb.
Perspect. Med. 2(12), a010272 (2012).
115 Warfel JM, Zimmerman LI, Merkel TJ.
Acellular pertussis vaccines protect against
disease but fail to prevent infection and
transmission in a nonhuman primate model.
Proc. Natl Acad. Sci. USA 111(2), 787–792
(2013).
116 Storsaeter J, Hallander HO, Gustafsson L,
Olin P. Levels of anti-pertussis antibodies
related to protection after household exposure
to Bordetella pertussis. Vaccine 16(20),
1907–1916 (1998).
117 Rappuoli R. The vaccine containing
recombinant pertussis toxin induces early and
long-lasting protection. Biologicals 27(2),
99–102 (1999).
118 Locht C, Mielcarek N. Live attenuated
vaccines against pertussis. Expert Rev. Vaccines
1, 1–12 (2014).
future science group