Am. J. Trop. Med. Hyg., 84(5), 2011, pp. 653–661
Copyright © 2011 by The American Society of Tropical Medicine and Hygiene
Leishmaniasis affects millions of people worldwide. It
includes a heterogeneous group of diseases that are caused by
protozoan parasites of the genus Leishmania . Zoonotic cuta-
neous leishmaniasis (ZCL) is widespread in central Tunisia
where it constitutes a public health problem with an annual
incidence of ~5,000 cases. 1 The clinical spectrum of the infec-
tion ranges from asymptomatic (sub-clinical) infection to
self-limited cutaneous sores or more severe disease result-
ing in disfiguring scars. The etiological agent is an Old World
Leishmania species, Leishmania major , which is transmitted
by the sand fly vector, Phlebotomus papatasi . Transmission is
greatest in the summer months (May to September) and the
development of cutaneous lesions tends to appear in humans
between October and March. 2
While probing for a blood meal, the infected sand flies sali-
vate into the host’s skin. Sand fly saliva contains potent vaso-
dilators, maxadilan and adenosine, described respectively in
Lutzomyia longipalpis and P. papatasi, that prevent clotting
at the biting site. 3, 4 Additionally, as clearly demonstrated by
several investigators, sand fly saliva contains immunomodu-
latory molecules that have been shown to enhance disease
progression. 5– 8
The possibility that leishmaniasis could be prevented by
vaccination against sand fly saliva was supported by previous
reports showing that pre-exposure to uninfected bites or pre-
immunization with saliva abolished the enhancing effect of
the saliva and/or prevented the disease in mice. 8– 10 Protection
could be conferred by the host’s antibodies that may neutral-
ize a yet unidentified immunomodulatory component in the
sand fly saliva. 8 Alternatively, other data suggested that the
protective effects of pre-exposure of saliva could be conferred
in mice by a cell-mediated immune response and a delayed-
type hypersensitivity to the salivary antigens. 9, 11, 12
Little is known about the antibody response against sand
fly saliva in humans. Individuals living in endemic areas for
leishmaniasis develop antibodies against the saliva of the
sand fly vectors 13– 15 ; yet, the protective role of these antibod-
ies remains controversial. A correlation between the intensity
of the antibody production against saliva of L. longipalpis and
the appearance of a cell-mediated and a protective immunity
against Leishmania chagasi has been shown. 14 Contrastingly,
the incidence of cutaneous leishmaniasis was still high in
endemic areas even though the inhabitants were frequently
bitten by uninfected sand flies arguing against a protective
effect of pre-exposure to saliva in humans. 15
To better understand the role of anti-sand fly saliva antibod-
ies in humans, we characterized the features of the humoral
immune response to the saliva of P. papatasi in 200 children
who live in an endemic area of ZCL in Tunisia and monitored
the antibody response throughout two seasons of Leishmania
MATERIAL AND METHODS
Ethics statement. All experiments were conducted accord-
ing to the principles expressed in the Declaration of Helsinki.
The study was approved by the ethic committee of the Institute
Pasteur of Tunis. All participants provided written informed
consent for the collection of blood samples and subsequent
Study population and samples. Two hundred children, part
from a previous study of ZCL in Tunisia, (age ranging from
6 to 12 years with a median of 8.2 years) were from endemic
areas in Central regions (El Guettar and Souk Ejjdid).
They were selected among 637 children on the basis of the
availability of serum samples at different time points of the
study. This cohort was followed up prospectively from April
2001 to October 2002 throughout two seasons of L. major
trans mission ( Figure 1 ). Blood samples were collected from
all donors at three different time points. The first sample was
obtained at the beginning of the study (April–May 2001) before
the ZCL transmission season, the second sample was collected
after the first transmission season (April–May 2002), and the
third sample was obtained in October 2002 immediately after
the second transmission season. Several parameters such as
leishmanin skin test (LST) reactivity and a complete clinical
examination in search of the presence of typical scars or
new active lesions were monitored during the study. Eighty-
two individuals tested positive for the leishmanin skin test at
Characterization of the Antibody Response to the Saliva of Phlebotomus papatasi in People
Living in Endemic Areas of Cutaneous Leishmaniasis
Soumaya Marzouki ,† Mélika Ben Ahmed ,* † Thouraya Boussoffara , Maha Abdeladhim , Nissaf Ben Aleya-Bouafif ,
Abdelkader Namane , Nabil Belhaj Hamida , Afif Ben Salah , and Hechmi Louzir
Department of Clinical Immunology, Institut Pasteur de Tunis, Tunisia; LIVGM, Institut Pasteur de Tunis, Tunisia; Department of Medical
Epidemiology, Institut Pasteur de Tunis, Tunisia; Institut Pasteur de Paris, Plate-forme de Protéomique, Paris, France
Abstract. Important data obtained in mice raise the possibility that immunization against the saliva of sand flies
could protect from leishmaniasis. Sand fly saliva stimulates the production of specific antibodies in individuals living in
endemic areas of parasite transmission. To characterize the humoral immune response against the saliva of Phlebotomus
papatasi in humans, we carried out a prospective study on 200 children living in areas of Leishmania major transmission.
We showed that 83% of donors carried anti-saliva IgG antibodies, primarily of IgG4 isotype. Positive sera reacted differ-
entially with seven salivary proteins. The protein PpSP30 was prominently recognized by all the sera. The salivary proteins
triggered the production of various antibody isotypes. Interestingly, the immunodominant PpSP30 was recognized by all
IgG subclasses, whereas PpSP12 was not by IgG4. Immunoproteomic analyses may help to identify the impact of each
salivary protein on the L. major infection and to select potential vaccine candidates.
* Address correspondence to Mélika Ben Ahmed, Laboratoire
d’Immunologie clinique, Institut Pasteur de Tunis, 13, Place Pasteur,
1002 Le Belvédère, Tunisia. E-mail: email@example.com
† These authors contributed equally to this work.
MARZOUKI AND OTHERS
enrollment and thirty five individuals developed ZCL after
the first transmission season (data not shown) .
Salivary glands extract preparation. Sand fly salivary glands
were kindly provided by Pr. E. Zhioua (Pasteur Institute of
Tunis). They were obtained from a colony of P. papatasi that
originated from El Felta, an endemic focus of ZCL located in
the governorate of Sidi Bouzid in Central Tunisia. 16 In some
experiments, salivary gland extracts (SGE) derived from a colony
of P. papatasi that originated from Anatolia, Turkey were used. 16
The glands were dissected out in cold Tris buffer (20 mM Tris,
150 mM NaCl pH = 7.6), and then disrupted by three freezing
and thawing cycles. After centrifugation, the supernatants were
stored at −80°C with 10% glycerol. The salivary gland extracts
were prepared just before use by dilution in phosphate saline
buffer (Invitrogen, Cergy Pontoise, France).
Serum IgG anti-SGE antibody detection. Specific anti-
saliva IgG antibodies were measured by enzyme-linked
immunosorbent assay (ELISA). The wells (NUNC, Maxisorp,
Roskilde, Denmark ) were coated overnight with SGE (0.5
glands per well) in 0.1 M carbonate-bicarbonate buffer (pH 9.6)
at 4°C. The wells were then washed in phosphate buffer (PBS)
with 0.1% Tween 20 and then incubated with 0.5% gelatin in
PBS buffer with 0.1% Tween 20 for 1 hour at 37°C to block
free binding sites. After washing, diluted sera (1:200) were
incubated for 2 hours at 37°C. Antibody-antigen complexes
were detected using peroxidase-conjugated anti-human IgG
antibody diluted at 1:10,000 (Sigma, St. Louis, MO) for 1 hour
at 37°C and visualized using orthophenylendiamine (OPD)
in citrate buffer and hydrogen peroxide. The absorbance was
measured using an automated ELISA reader (Awareness
Technology Inc., Palm City, FL) at 492-nm wavelength. Because
sand flies are present in almost all the country, 20 negative sera
were obtained from healthy controls living outside Tunisia
in sand fly-free regions. The cut-off value for the assays was
the mean optical density (OD) of 20 negative controls plus
three standard deviations. The relative OD was defined as
the ratio of sample OD/mean OD of sera from 20 negative
Detection of serum IgG subclasses (IgG1, 2, 3, and 4) and IgE
anti-SGE antibody. Specific anti-saliva IgG1, IgG2, IgG3, IgG4,
and IgE antibodies were measured by ELISA. The optimal
conditions of antigen concentration as well as sample, primary
antibody and streptavidine-horseradish peroxidase dilutions
were previously determined based on the differences obtained
by positive and negative sera. The wells were coated with SGE
and free binding sites were blocked as described previously.
The wells were then incubated with diluted serum samples
(1:200) for 2 hours at room temperature. After six washes,
biotin-conjugated anti-human IgG isotypes (Sigma) or IgE
(BD Biosciences, Le Pont de Claix, France) were incubated for
1 hour at 37°C at a dilution of 1:2,000; 1:20,000; 1:4,000; 1:20,000,
or 1:250 for IgG1, IgG2, IgG3, IgG4 and IgE, respectively. After
eight washes, streptavidine-horseradish peroxidase diluted at
1:6,000 (Amersham, Little Chalfont Buckinghamshire, UK)
was added for 45 minutes at 37°C. Antibody-antigen complexes
were visualized using OPD in citrate buffer and hydrogen
peroxide. The absorbance was measured at 492 nm wavelength.
The cut-off for the assays was the mean OD of 20 negative
controls plus three standard deviations.
Western-blot analysis. Salivary gland extracts were sepa-
rated on a 12% or 15% sodium dodecyl sulfate- polyacrylam-
ide gel electrophoresis (SDS-PAGE) . The optimal conditions
of antigen concentration as well as sample, primary antibody
and streptavidine-horseradish peroxidase dilutions were pre-
viously determined. The equivalent of 40–60 salivary glands
was loaded into a single long-well. The separated proteins were
then transferred onto a nitrocellulose membrane. The mem-
brane was incubated overnight at 4°C with a blocking buffer
containing 5% nonfat milk and then cut into 8 to 10 strips.
Each strip was incubated for 1 hour at room temperature with
serum samples at different dilutions for each assay (1:200 for
IgG1, IgG2, and IgG4, 1:20 for IgE and IgG3). After washing,
the strips were incubated with horseradish peroxidase-linked
anti-human IgG antibody (Sigma) at 1:10,000 or biotin-
conjugated anti-human IgG1, IgG2, IgG3, IgG4 (Sigma) at
different dilutions (1:1,000; 1:300; 1:500; 1:5,000, respectively)
or IgE (BD Biosciences) at 1:250 for 1 hour at room tempera-
ture. A 45-minute incubation step at room temperature with
streptavidine-horseradish peroxidase at 1:10,000 (Amersham)
was performed when biotin-conjugated anti-human antibod-
ies were used. After five washings, positive bands were visu-
alized using enhanced chemiluminescence (Amersham). For
all experiments, different molecular weight markers were used
(RPN 800 or RPN 800E, Amersham).
Identification of salivary proteins by mass spectrometry
(MS). Salivary gland extracts of a Tunisian strain of P. papatasi
Figure 1. Timeline showing the temporal relationship between key events in the study and two seasons of Leishmania major transmission.
Two hundred participants living in endemic areas of zoonotic cutaneous leishmaniasis (ZCL) in Central and Southwestern Tunisia (Gafsa and Sidi
Bouzid) were followed up over 2 years throughout two seasons of L. major transmission. Several parameters such as leishmanin skin test or the
presence of typical scars were monitored at the beginning of the study and after each transmission season and the triggering of new cases. Peripheral
blood samples were obtained from each donor during these examinations.
HUMAN ANTIBODY RESPONSE AGAINST SAND FLY SALIVA
were fractioned in a 15% SDS-PAGE gel. After Coomassie
staining, gel bands corresponding to the major proteins that
were recognized by positive sera were excised using the
ProPic Investigator (Genomic Solutions, Ann Arbor, MI) and
collected into a 96-well plate. Distaining, reduction, alkylation,
and trypsin digestion of the proteins followed by peptide
extraction were carried out with the ProGest Investigator
For MS and MS/MS analysis, peptides were eluted after
the desalting step (C18-μZipTip, Millipore, Billerica, MA )
directly using the ProMS Investigator, (Genomic Solutions)
onto a 96-well stainless steel MALDI target plate (Applied
Biosystems/MDS SCIEX, Framingham, MA) with 0.5 μL of
CHCA matrix (5 mg/mL in 70% ACN/30% H 2 O/0.1% TFA).
Raw data for protein identification were obtained on the
4800 Proteomics Analyzer (Applied Biosystems) and analyzed
with the GPS Explorer 3.6 software (Applied Biosystems). For
positive-ion reflector mode spectra 3,000 laser shots were aver-
aged. For MS calibration, autolysis peaks of trypsin ([M+H] + =
842.5100 and 2211.1046) were used as internal calibrates.
Monoisotopic peak masses were automatically determined
within the mass range 800–4000 Da with a signal to noise ratio
minimum set to 30. Up to 20 of the most intense ion signals
were selected as precursors for MS/MS acquisition exclud-
ing common trypsin autolysis peaks and matrix ion signals. In
MS/MS positive ion mode, 4,000 spectra were averaged, col-
lision energy was 2 kV, collision gas was air, and default cali-
bration was set using the Glu 1 -Fibrino-peptide B ([M+H] + =
1570.6696) spotted onto 14 positions of the MALDI target.
Combined peptide mass fingerprint (PMF ) and MS/MS que-
ries were performed using the MASCOT search engine 2.1
(Matrix Science Ltd., London, UK) embedded into GPS-
Explorer Software 3.6 (Applied Biosystems) on the National
Center for Biotechnology Information (NCBI ) database
(downloaded 2008 10 22; 7135729 sequences, 246233216 resi-
dues) with the following parameter settings: 50-ppm peptide
mass accuracy, trypsin cleavage, one missed cleavage allowed,
carbamidomethylation set as fixed modification, oxidation of
methionines was allowed as variable modification. The MS/
MS fragment tolerance was set to 0.3 Da. Only peptides with
a MASCOT Ion score ≥ 51 (GPS Explorer confidence index ≥
99%) were taken in account for protein identification. Protein
hits with MASCOT Protein score ≥ 81 and a GPS Explorer
Protein confidence index ≥ 99% were used for further manual
Statistical analysis. The variation in the percentage of
positive donors and the median of the relative OD of anti-
SGE antibodies throughout two transmission seasons was
analyzed by McNemar’s and Dunnet tests, respectively. The
levels of serum IgG and IgE antibodies in donors with and
without leishmaniasis were compared by using the non-
parametric Mann-Whitney U test. The correlation between
the levels of different isotypes of antibodies was assessed
using Spearman’s rank correlation. Statistical significance was
assigned to a value of P < 0.05.
Follow-up of IgG antibodies produced in response to
the saliva of P. papatasi in a cohort of children living in an
endemic area of L. major transmission. The antibody response
of 200 children was studied over 2 years corresponding to two
transmission seasons of L. major . As shown in Figure 2 , 83%
of the participants tested positive for IgG antibodies against
Figure 2. Serum levels of IgG antibodies to Phlebotomus papatasi saliva in people living in endemic areas of Leishmania major transmission.
IgG antibodies to salivary gland extracts from P. papatasi were investigated in 200 children who live in endemic areas of zoonotic cutaneous leish-
maniasis (ZCL). The IgG antibody levels were measured at three sampling points; at the beginning of the study before the transmission season
(sample 1) and after one transmission season (sample 2) and two transmission seasons (sample 3). ( A ) The percentage of participants who had IgG
antibodies to P. papatasi saliva did not vary significantly throughout the two transmission seasons ( P > 0.05). ( B ) The results are expressed as the
relative optical density (OD), which was the ratio of sample OD/mean OD of sera from 20 negative controls. The threshold (cut-off for the assays)
was the mean optical density of sera from 20 negative controls plus three standard deviations. ( C ) Diagram showing the follow-up of the IgG anti-
saliva antibodies throughout the three sampling points in the sera of all donors. Only three donors tested negative for specific IgG antibodies
throughout the three sampling points. ( D ) Serum levels of IgG anti-saliva antibodies in 26 individuals with a naive immunological status against
L. major. Of these 26 participants, 10 individuals developed ZCL after one transmission season (ZCL [+]) and 16 did not (ZCL [−]). The upper,
middle, and lower box lines represent the 75th percentile, the median, and the 25th percentile of the OD values obtained in each group. The statisti-
cal significance between the two groups is based on the Mann-Whitney test.
MARZOUKI AND OTHERS
sand fly saliva at the beginning of the study. Although the
median of the relative OD anti-SGE IgG antibodies varied
throughout the seasons, the percentage of positive donors did
not change significantly ( Figure 2A and B). Among the 165
children who tested positive for IgG anti-saliva antibodies at
the beginning of the study, 126 remained positive throughout
the study and 39 donors became negative in the second and/or
third sampling time ( Figure 2C ). Conversely, of the 35 donors
who tested negative for anti-saliva IgG antibodies in the first
blood sample, 27 developed specific anti-saliva antibodies
at the second sampling and five tested positive at the third
sampling. Only three children (9% of negative cases) remained
unresponsive after two transmission seasons ( Figure 2C ).
Altogether, these data suggest that nearly 90% of children
living in an endemic area of ZCL who test negative for IgG
anti-saliva antibodies develop specific antibodies within two
We (H. Louzir, IPSOS V) and others 17 formerly showed that
the presence of salivary antibodies constitutes a risk factor
for the development of ZCL. We, thus, focused our analysis
on children who were negative for LST at the beginning of
our study and who did not have any clinical evidence of
During our follow-up, we found that the presence of IgG
antibodies against saliva in this subgroup was associated with
an increased risk of ZCL. Indeed, 9 of 20 (45%) children with
anti-sand fly antibodies developed ZCL, whereas only 1 of
6 (16%) donors without antibodies contracted the disease
(data not shown). Furthermore, the median level of the anti-
saliva antibodies was significantly greater in patients who later
developed ZCL ( P = 0.03) ( Figure 2D ).
Characteristics of the antibodies to P. papatasi saliva in
humans. To better define the features of the IgG antibodies
directed against the saliva of sand flies, we analyzed by
an ELISA test the subclasses of these antibodies in 65
representative positive sera that cover the different range of
positivity. Although IgG4 is a minor isotype among the IgG
antibodies, anti-saliva antibodies were prominently of IgG4
subclass (76%) and at a lesser extent of IgG2 (50%) or IgG1
(39%) and IgG3 (39%) isotypes ( Figure 3 , panels A and B).
Furthermore, the relative OD of IgG4 anti-saliva antibodies
were significantly higher than those of the other isotypes
Figure 3. Quantification of IgG subclasses and IgE anti-saliva antibodies. Levels of IgG1, IgG2, IgG3, IgG4, and IgE antibodies directed against
Phlebotomus papatasi saliva were studied in serum samples of 65 participants with specific IgG antibodies. ( A ) The percentage of positive donors
was calculated for each assay. The results are expressed for ( B ) IgG subclass and ( C ) IgE antibodies as the relative optical density (OD), which cor-
responds to the ratio of sample OD/mean OD from the sera of 20 negative controls. The threshold corresponds to the mean OD obtained with the
sera of 20 negative controls plus three standard deviations. ( D ) Regression line for pairs of OD values corresponding to IgG4 and IgE anti-saliva
antibodies is shown for the 65 IgG-positive participants. No correlation between the levels of IgG4 and IgE anti-saliva antibodies was found. The
Spearman’s rank correlation coefficient (rho) and the P value are indicated. ( E ) The serum levels of IgE anti-saliva antibodies in 26 individuals with
a naive immunological status against Leishmania major are shown. Of these 26 participants, 10 individuals developed ZCL after one transmission
season (ZCL [+]) and 16 did not (ZCL [−]). The upper, middle, and lower box lines represent the 75th percentile, the median, and the 25th percentile
of the OD values obtained in each group. The statistical significance between the two groups is based on the Mann-Whitney test.
HUMAN ANTIBODY RESPONSE AGAINST SAND FLY SALIVA
( Figure 3A ). Interestingly, the level of IgG4 anti-saliva
antibodies correlated positively with the level of specific IgG1
and IgG2 antibodies ( P = 0.01 and P = 0.09, respectively) but
not with specific IgG3 antibodies ( P > 0.05) (data not shown).
The IgE antibodies against mosquito saliva are commonly
observed in the sera of people exposed to mosquito bites. 18, 19
They may indicate a classic type I hypersensitivity 20 and are
often associated with IgG4 antibodies. 18 To test whether the
presence of IgG4 antibodies against P. papatasi saliva anti-
bodies was related or not to an allergic reaction to the saliva,
the production of IgE antibodies against P. papatasi saliva
was investigated and the correlation between IgE and IgG4
antibodies analyzed. Although 44% of children with posi-
tive IgG antibodies to saliva had also specific IgE antibodies
( Figure 3C ), there was no correlation ( P = 0.43) between the
presence of IgG4 and IgE antibodies ( Figure 3D ). This sug-
gests that IgG4 production is rather a specific feature of the
immune response against P. papatasi saliva and not an aller-
gic phenomenon. Interestingly, the presence of IgE anti-saliva
antibodies was associated with an enhanced risk of triggering
ZCL among the 26 children who lacked clinical and biological
features of previous ZCL at enrollment. Indeed, 7 of 10 (70%)
of children with IgE antibodies developed ZCL, whereas
only 3 of 16 (18%) of those without specific IgE antibodies
acquired the disease. Furthermore, the median level of the IgE
antibodies was significantly higher in patients who later devel-
oped ZCL ( P = 0.005) ( Figure 3E ).
Identification of the target proteins in P. papatasi saliva.
The Western-blot analysis showed that seven salivary proteins
of 12kDa, 15kDa, 21kDa, 28kDa, 30kDa, 36kDa, and 44kDa
were commonly, but differentially, recognized by the tested
positive sera ( Figure 4A ). The frequency and intensity of
reactivity against these different proteins were evaluated in
20 positive sera selected randomly from donors with anti-
saliva IgG antibodies ( Table 1 ). To better distinguish between
the reactivity against the 12kDa and 15kDa proteins, we
reanalyzed some sera in blots after a 15% SDS-PAGE (data
not shown). The 30kDa protein was prominently recognized by
all these sera, whereas the other proteins were only moderately
and inconsistently recognized ( Table 1 ). Nearly 90% of the
positive sera showed a moderate recognition of the 12kDa,
15kDa, and 36kDa proteins, whereas the reactivity against the
21kDa, 28kDa, and 44kDa proteins was only lightly detected
in 60–70% of the positive sera ( Table 1 ).
The target proteins in the saliva of P. papatasi were identified
by MS after trypsin digestion and elution ( Table 2 ). As seen in
Figure 4B , six of the target proteins correspond to previously
identified salivary proteins in P. papatasi —PpSP12 (SL1 pro-
tein, gi|15963505); PpSP15 (SL1 protein, gi|15963509); PpSP28
(D7 protein, gi|15963513); PpSP30(D7 protein, gi|15963513);
PpSP36 (Salivary apyrase, gi|10443907); PpSP44 (yellow pro-
tein, gi|15963519). The 21kDa protein was difficult to iden-
tify because it was present at such a low level. However, a
three-dimensional analysis of the Coomassie-stained SDS
gel revealed a light expression of this protein in a wide area
between PpSP15 and PpSP28 ( Figure 4C ). We subsequently
enlarged the size of the band cut and a new MS analysis
revealed that the 21kDa target protein matched the salivary
protein PpSP42 (yellow protein, gi|15963517).
Finally, we showed that the immunodominant PpSP30
protein was recognized by all sub-classes of IgG antibodies
( Figure 5 ). The PpSP15 and PpSP36 were recognized mostly
by IgG1, IgG2, and IgG4 antibodies, whereas PpSP12 protein
was targeted mainly by IgG1 and more inconsistently by IgG2
Figure 4. Identification of the target proteins in the saliva of Phlebotomus papatasi . Salivary gland extracts were fractionated with a ( A ) 12%
or ( B , C ) 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). ( A ) Immunoblots profiles of salivary antigens from a
Tunisian strain of P. papatasi with positive (1–7) and negative (−) human sera are shown. The molecular weight marker is shown. ( B ) A Coomassie-
stained SDS-PAGE gel of 20 homogenized pairs of salivary glands from P. papatasi is shown in Lane 2. Lane 1 shows the molecular weight marker
(RPN 800E, Amersham), whereas lane 3 shows the corresponding blot with a positive tested serum. ( C ) A three-dimensional analysis of the subre-
gion of Coomassie-stained gel (shown in the upper panel) using PD Quest software.
MARZOUKI AND OTHERS
antibodies but not by the other isotypes. In positive sera, IgE
antibodies against SGE were directed mainly against PpSP30
and the 21kDa protein.
Phlebotomus papatasi is one of the major sand fly vectors of
Leishmania in the Old World. A spatial correlation between the
abundance of P. papatasi in Tunisia and the incidence of ZCL
showed that the disease is endemic in the arid and Saharan
bioclimatic areas corresponding to Central and Southwestern
Tunisia. 21 Little is known about the human antibody response
to the bites of sand flies from the Old World. In Sanliurfa,
Turkey (a focus of Leishmania tropica ), Rohousova and others 15
showed that ~40% of the people carried IgG antibodies to the
saliva of P. papatasi . In this study, nearly 80% of our participants
tested positive, a result that may suggest a higher exposure to
P. papatasi bites in the selected areas. Because there was no infor-
mation about the age of the participants tested by Rohousova
and others, such discrepancy could also be explained by a dif-
ference in the median age of the two cohorts .
Monitoring the antibody response to sand fly saliva revealed
that nearly 90% of children who tested negative for IgG anti-
bodies against P. papatasi saliva developed specific antibodies
after two transmission seasons. However, the overall percent-
age of positive children did not vary significantly through-
out the time. Interestingly, most of the IgG antibodies were
of the IgG4 isotype and to a lesser extent, of the IgG2 sub-
class. One published study analyzed the IgG isotypes devel-
oped by humans in response to the sand fly saliva. It reported
that individuals who were experimentally exposed to the bites
of uninfected L. longipalpis developed anti-saliva antibodies
restricted mostly to IgG1 and IgG4 subclasses. 22 Such results
were close to those obtained in individuals exposed to the
bites of Aedes . 23– 26 In all these studies, the presence of IgG4
antibodies was correlated to the production of IgE antibodies
suggesting that IgG4 was the result of an allergic reaction to
mosquitoe bites. Contrasting with such data, we did not find
any correlation between the presence of specific IgG4 and IgE
antibodies in our cohort suggesting that the prominence of
IgG4 anti-saliva antibodies did not reflect an allergic response
(hypersensitivity type I) to sand fly bites. Moreover, the IgE
antibodies were restricted to two salivary antigens, whereas
the IgG4 antibodies recognized all the target proteins.
In light of these results, one might expect that the recall of a
memory immune response in previously sensitized individuals
Features of salivary protein recognition with positive sera
Salivary proteins *
12 kDA 15 kDA 21 kDA 28 kDA 30 kDA 36 kDA 44 kDA
Percentage of positive sera
Intensity of reaction †
94.1% 88.1% 70.5% 60.5% 100% 94.1%64.7%
* The percentage of positive sera and the intensity of the reaction were determined by western-blotting in 20 donors selected randomly from those with IgG anti-SGE.
† The intensity of reaction was quoted 0 (absence), 1 (fair reactivity), 2 (moderate reactivity), 3 (strong reactivity) or 4 (very strong reactivity). The mean intensity represents the average intensity
of reaction of each protein obtained in the 20 positive sera.
Protein identification of antibody target proteins by mass spectrometry
bands (kDa)Protein name
coverage (%)Peptide sequences identified by MS/MS experiment
36 salivary apyrase
gi|10443907 38.3 1861747
* NCBI = National Center for Biotechnology Information; MW = molecular weight; MS = mass spectrometry; MASCOT = search engine.
HUMAN ANTIBODY RESPONSE AGAINST SAND FLY SALIVA
would impair the host response and lead to the commitment
of the anti- Leishmania immunity toward a Th2 response. This
response may support an exacerbating effect of pre-exposure
to sand fly saliva in humans. People living in endemic areas
are normally exposed to bites of wild populations of P. papa-
tasi . In Tunisia, the highest prevalence of infection of P. pap-
atasi with L. major during the transmission peak is 7.9%. 2, 27
The high seroprevalence observed among our study popula-
tion is the result of a high degree of contact between people
and wild populations of P. papatasi . Therefore, despite that
pre-exposure to uninfected bites of P. papatasi are more fre-
quent than infected ones, people are still succumbing to ZCL
in endemic areas. This finding is corroborated by the results
of a recent study showing that pre-immunization of mice with
salivary gland proteins of wild-caught Tunisian strain of P. pap-
atasi did not provide any protection against L. major infection
compared with a significant protection obtained with salivary
gland proteins of long-term colonized ones. 28 The absence of
protection is probably caused by the polymorphism of salivary
gland proteins of the wild population of sand flies to avoid
host immune systems. 28, 29 Interestingly, when we followed up
the participants with a naive immunological status against
L. major, we showed that children with anti-saliva antibodies
were more likely to develop ZCL. Accordingly, the levels of
anti-saliva IgG antibodies were significantly higher in patients
who developed ZCL. Our results are consistent with the data
from two previous reports on leishmaniaisis from either the
New World 16 or the Old Word (Louzi H, personal communica-
tion). More generally, a correlation between the level of spe-
cific IgG anti-saliva antibodies and the risk of disease has been
demonstrated for different vector-host models. 30– 32 We also
found that the enhanced risk of developing ZCL was associ-
ated with the production of anti-saliva antibodies of IgE iso-
type. One may suggest that individuals who have allergies are
more prone to develop a Th2 immune response and therefore
have an increased risk of developing leishmaniasis.
Interestingly, our parallel work on the cellular immune
response developed against P. papatasi saliva further con-
firms that this immune response is dominated by a Th2 profile.
Indeed, we showed that specific memory cells are mainly CD8 +
T lymphocytes that produce high levels of Interleukin-10
(IL-10) and IL-4 (Abdeladhim and others, manuscript in
preparation ). Nevertheless, we also showed that specific IFN-
γ-producing CD4 + T cells could be activated after block-
ing IL-10 production (Abdeladhim and others, manuscript
in preparation). This fact is of great interest as it provides a
new rationale for immunological approaches that target the
salivary components inducing a Th1 immune response. This
strategy could be used to create a combined vaccine that con-
tains Leishmania antigens and salivary components. For that
purpose, we further investigated the proteins from P. papatasi
saliva that are targeted by the antibody response. In accordance
with previous results obtained by Rohousova and others, 15 we
found that all positive sera from naturally exposed children
reacted strongly with a 30kDa protein, suggesting that this
protein is the major immunodominant antigen of P. papatasi
saliva in humans. Clements and others 33 recently highlighted
the utility of measuring the antibody response to the saliva of
Phlebotomus argentipes to assess the exposure among humans
living in endemic areas for leishmaniasis and subsequently
to evaluate vector control programs. However, these authors
showed the possibility of cross-reactive reactions between dif-
ferent species of sand flies. Thus, developing serological tests
with the recombinant form of the immunodominant protein
may overcome such limits.
Other salivary antigens such as 12 kDa, 15 kDa, and 36 kDa
proteins were also frequently recognized by positive sera but
to a lesser degree. Noticeably, the 12 kDa and 15 kDa pro-
teins have not been demonstrated by Rohousova and others 15
as target antigens. Previous studies showed that the compo-
sition and antigenicity of sand fly saliva may vary with the
geographical origin of the fly. 34 Because the Anatolian strain
differs from the Tunisian strain in that it can only reproduce
anautogenously, needs a previous blood meal to produce fer-
tile eggs, 16 we tested the reactivity of our positive sera against
the saliva of a P. papatasi strain that originated from Anatolia
in Turkey but no significant difference in the reactivity pattern
between the two tested strains of P. papatasi was found (data
Mass spectrometry analyses showed that the immunodom-
inant salivary protein recognized by all the tested sera was
related to PpSP30 and the sequence obtained from the cor-
responding band was different from that obtained from the
bands of 28 kDa and 32 kDa. Similarly, five of the other six
target proteins have been previously described and corre-
spond to PpSP12, PpSP15, PpSP28, PpSP36, and PpSP44. 12
Regarding the target protein of a 21 kDa antigen, which has
Figure 5. IgG subclasses and IgE immunoblots against sali-
vary proteins of Phlebotomus papatasi . Salivary gland proteins of
P. papatasi were fractioned with a 15% sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDS-PAGE). IgG subclasses
and IgE antibodies react against salivary antigens from P. papatasi in
three positive participants (1–3). Immunoblots of one negative human
serum is also shown. The results were representative of 6 positive sera
for IgG subclasses.
MARZOUKI AND OTHERS
not been previously described, further analyses revealed that
it was related to the yellow protein PpSP42, corresponding
possibly to a minor isoform of this protein. However, we can-
not exclude that 21 kDa antigen might be a product of deg-
radation or proteolytic cleavage of the PpSP42. Remarkably,
PpSP42 exhibits much homology with PpSP44, another yellow
protein that we identified as a target protein of antibodies in
humans. Further analysis of the identity of the salivary pro-
teins of P. papatasi using proteomic approaches are in progress
and may bring some clarification to this issue.
Finally, our data showed that the subclasses of IgG anti-
bodies to the saliva of P. papatasi may differ according to the
target protein. Although PpSP15, PpSP30, and PpSP36 were
recognized by all IgG isotypes, PpSP12 was targeted only by
IgG1 and IgG2 antibodies and not by IgG4 and IgE antibod-
ies. This suggests that the immune response mediated by this
antigen may not be polarized toward a Th2 phenotype. We
are currently investigating the immune profiles induced by
the different salivary gland proteins and attempting to predict
their specific effects on the outcome of leishmaniasis in a large
cohort of naturally exposed individuals. These investigations
could help us to define the salivary proteins that might be use-
ful for vaccination against leishmaniasis.
In conclusion, our data suggest that antibody response
developed by naturally exposed individuals against saliva of P.
papatasi is mainly polarized toward a Th2 phenotype. This anti-
body response is mainly directed toward an immunodominant
protein, PpSP30. The production of the related recombinant
protein and the confirmation of its recognition by the posi-
tive sera are currently in progress. Such experiments will serve
ultimately to develop a serological test that could be useful for
monitoring exposure of humans to sand fly bites and for pre-
dicting the risk of triggering leishmaniasis. Additionally, immu-
noproteomic analyses of target proteins may help us to better
define the impact of each protein on Leishmania infection and
possibly to define potential candidates for vaccination.
Received October 21, 2010. Accepted for publication January 15,
Acknowledgments: We thank E. Zhioua and I. Chelbi for the critical
review of the manuscript. We also thank the International Clinical
Sciences Support Center (ICSSC) at Family Health International for
editorial support as we prepared the manuscript.
Financial support: This work was supported by the NIH/NIAID grant
Authors’ addresses: Soumaya Marzouki, Mélika Ben Ahmed, Maha
Abdeladhim, and Hechmi Louzir, Laboratoire d’Immunologie clin-
ique, Institut Pasteur de Tunis, Tunisia, E-mails: marzouki_sou@yahoo
.fr , firstname.lastname@example.org , email@example.com , and
firstname.lastname@example.org . Thouraya Boussoffara, Laboratoire
d’Immunologie, de Vaccinologie et de Génétique Moléculaire, Institut
Pasteur de Tunis, Tunisia, E-mail: email@example.com
.tn . Nissaf Ben Aleya-Bouafif, Nabil Belhaj Hamida, and Afif Ben
Salah, Laboratoire d’Épidémiologie Médicale, Institut Pasteur de
Tunis, Tunisia, E-mails: firstname.lastname@example.org , nabil.belhadjhmida@
pasteur.rns.tn , and email@example.com . Abdelkader Namane,
Plateforme de Protéomique, Institut Pasteur de Paris, Paris, France,
E-mail: firstname.lastname@example.org .
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