Immunodominant antigens of Leishmania chagasi associated with protection against human visceral leishmaniasis.
ABSTRACT Protection and recovery from visceral leishmaniasis (VL) have been associated with cell-mediated immune (CMI) responses, whereas no protective role has been attributed to humoral responses against specific parasitic antigens. In this report, we compared carefully selected groups of individuals with distinct responses to Leishmania chagasi to explore antigen-recognizing IgG present in resistant individuals.
VL patients with negative delayed-type hypersensitivity (DTH) were classified into the susceptible group. Individuals who had recovered from VL and converted to a DTH+ response, as well as asymptomatic infected individuals (DTH+), were categorized into the resistant group. Sera from these groups were used to detect antigens from L. chagasi by conventional and 2D Western blot assays. Despite an overall reduction in the reactivity of several proteins after DTH conversion, a specific group of proteins (approximately 110-130 kDa) consistently reacted with sera from DTH converters. Other antigens that specifically reacted with sera from DTH+ individuals were isolated and tandem mass spectrometry followed by database query with the protein search engine MASCO were used to identify antigens. The serological properties of recombinant version of the selected antigens were tested by ELISA. Sera from asymptomatic infected people (DTH+) reacted more strongly with a mixture of selected recombinant antigens than with total soluble Leishmania antigen (SLA), with less cross-reactivity against Chagas disease patients' sera.
Our results are the first evidence of leishmania proteins that are specifically recognized by sera from individuals who are putatively resistant to VL. In addition, these data highlight the possibility of using specific proteins in serological tests for the identification of asymptomatic infected individuals.
- SourceAvailable from: Julio Scharfstein[Show abstract] [Hide abstract]
ABSTRACT: BACKGROUND: Kinins liberated from plasma--borne kininogens, are potent innate stimulatory signals. We evaluated whether resistance to infection by Leishmania (L.) chagasi depends on activation of G-protein coupled bradykinin B2 receptors (B2R). FINDINGS: B2R -/- C57BL/6 knock-out (KOB2) and B2R+/+ C57BL/6-wild type control mice (C57) were infected with amastigotes of Leishmania (L.) chagasi. Thirty days after infection, the KOB2 mice showed 14% and 32% relative increases of liver (p< 0.017) and spleen weights (p<0.050), respectively, whereas liver parasite load increased 65% (p< 0.011) in relation to wild type mice. The relative weight increases of liver and spleen and the parasite load were positively correlated (R = 0.6911; p< 0.007 to R = 0.7629; p< 0.001, respectively). Conversely, we found a negative correlation between the increased liver relative weight and the weakened DTH response (a strong correlate to protection or natural resistance to VL) or the decreased levels of IgGb antibodies to leishmanial antigen. Finally, we also found that IFN-gamma secretion by splenocytes, an adaptive response that was significantly decreased in KOB2 mice (p< 0.002), was (i) negatively correlated to the increase in liver LDU (R = -0.6684; p = 0.035) and liver/body relative weight (R = -0.6946; p = 0.026) and (ii) positively correlated to serum IgG2b levels (R = 0.8817; p = 0.001). CONCLUSIONS: We found that mice lacking B2R display increased susceptibility to the infection by Leishmania (L.) chagasi. Our findings suggest that activation of the bradykinin/B2R pathway contributes to development of host resistance to visceral leishmaniasis.Parasites & Vectors 11/2012; 5(1):261. · 3.25 Impact Factor
Immunodominant Antigens of Leishmania chagasi
Associated with Protection against Human Visceral
Daniel R. Aba ´nades1, Leonardo V. Arruda1,2, Elaine S. Arruda1, Jose ´ Roberto A. S. Pinto3, Mario S. Palma3,
Dorlene Aquino4, Arlene J. Caldas4, Manuel Soto5, Aldina Barral1,2,6, Manoel Barral-Netto1,2,6*
1Centro de Pesquisas Gonc ¸alo Moniz (CPqGM), Fundac ¸a ˜o Oswaldo Cruz (FIOCRUZ), Salvador, Bahia, Brazil, 2Faculdade de Medicina da Bahia, Universidade Federal da
Bahia, Salvador, Brazil, 3Center of the Study of Social Insects, Institute of Biosciences of Rio Claro, Department of Biology, University of Sa ˜o Paulo State (UNESP), Rio Claro,
Sa ˜o Paulo, Brazil, 4Departamento de Enfermagem, Universidade Federal do Maranha ˜o, Sa ˜o Luis, Maranha ˜o, Brazil, 5Centro de Biologı ´a Molecular Severo Ochoa (CSIC-
UAM), Departamento de Biologı ´a Molecular, Universidad Auto ´noma de Madrid, Madrid, Spain, 6Instituto Nacional de Cie ˆncia e Tecnologia de Investigac ¸a ˜o em Imunologia
(iii-INCT), Salvador, Bahia, Brazil
Background: Protection and recovery from visceral leishmaniasis (VL) have been associated with cell-mediated immune
(CMI) responses, whereas no protective role has been attributed to humoral responses against specific parasitic antigens. In
this report, we compared carefully selected groups of individuals with distinct responses to Leishmania chagasi to explore
antigen-recognizing IgG present in resistant individuals.
Methodology and Principal Findings: VL patients with negative delayed-type hypersensitivity (DTH) were classified into the
susceptible group. Individuals who had recovered from VL and converted to a DTH+ response, as well as asymptomatic
infected individuals (DTH+), were categorized into the resistant group. Sera from these groups were used to detect antigens
from L. chagasi by conventional and 2D Western blot assays. Despite an overall reduction in the reactivity of several proteins
after DTH conversion, a specific group of proteins (approximately 110–130 kDa) consistently reacted with sera from DTH
converters. Other antigens that specifically reacted with sera from DTH+ individuals were isolated and tandem mass
spectrometry followed by database query with the protein search engine MASCO were used to identify antigens. The
serological properties of recombinant version of the selected antigens were tested by ELISA. Sera from asymptomatic
infected people (DTH+) reacted more strongly with a mixture of selected recombinant antigens than with total soluble
Leishmania antigen (SLA), with less cross-reactivity against Chagas disease patients’ sera.
Significance: Our results are the first evidence of leishmania proteins that are specifically recognized by sera from
individuals who are putatively resistant to VL. In addition, these data highlight the possibility of using specific proteins in
serological tests for the identification of asymptomatic infected individuals.
Citation: Aba ´nades DR, Arruda LV, Arruda ES, Pinto JRAS, Palma MS, et al. (2012) Immunodominant Antigens of Leishmania chagasi Associated with Protection
against Human Visceral Leishmaniasis. PLoS Negl Trop Dis 6(6): e1687. doi:10.1371/journal.pntd.0001687
Editor: Edgar M. Carvalho, Hospital Universita ´rio, Brazil
Received January 4, 2012; Accepted April 30, 2012; Published June 19, 2012
Copyright: ? 2012 Aba ´nades et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The research and publication process was supported by grants from FAPESB: PES 0074/2008 Projetos Estrate ´gicos, from whom DRA and EA received
fellowships, and from Pronex: CNPq/FAPESB. MSP, AB, JC and MB-N are senior investigators from CNPq. The funders had no role in study design, data collection
and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
Visceral Leishmaniasis (VL) is a potentially fatal disease caused
by infection with Leishmania chagasi in the New World and
Leishmania donovani or Leishmania infantum in the Old World .
Infection leads to a spectrum of clinical outcomes ranging from
asymptomatic infection to active disease. The anti-Leishmania
immune response during asymptomatic infection is characterized
by a low serological and positive cellular response, which is
demonstrated by a positive delayed-type hypersensitivity skin
response (DTH+) . Patients with active VL, on the other hand,
present a strong positive serological and a negative cell-mediated
immune (CMI) response with low IFN-c production . Treated
patients that recover from illness (accounts for 90%) only exhibit a
positive DTH response long after treatment . Epidemiological
studies in Brazil showed that a positive DTH response is a marker
of protection against VL .
Serological diagnosis of L. chagasi during asymptomatic infection
is complicated by low antibody titers and frequent cross-reactivity
with other diseases . In addition, serologic markers of recovery
or resistance to infection have not been characterized. There is no
effective and safe vaccine approved for human use against any
form of visceral leishmaniasis despite the obvious need and
considerable effort that has been made.
In the present report, we compared the reactivity against total
protein extracts from L. chagasi of sera obtained from either DTH
positive patients (asymptomatic or treated and recovered individ-
uals) or symptomatic patients. A serological pattern associated with
www.plosntds.org1 June 2012 | Volume 6 | Issue 6 | e1687
DTH positivity was observed in both asymptomatic individuals
and in recovered patients. In addition, the recombinant version of
select antigens appeared to be a valuable tool for the serological
identification of asymptomatic patients.
Materials and Methods
This study was approved by the Research Ethics Committee of
the Federal University of Maranha ˜o University Hospital, Brazil
(localities where the field study was performed). All clinical
investigations were conducted according to the Declaration of
Helsinki. Written, informed consent was obtained from all
participants or legal guardians.
Leishmania chagasi (MHOM/BR00/MER/STRAIN2) promasti-
gotes were cultured in Schneider’s medium supplemented with
10% inactive FBS, 2 mM L-glutamine, 100 U/mL penicillin, and
100 mL/mL streptomycin.
Patients, sera and delayed-type hypersensitivity reaction
Sera were obtained at two distinct settings as described below:
The patients were classified as VL (pre-treatment, samples
obtained during active disease previous to treatment) and post-
treatment (samples from treated and cured patients). All patients
were from the Maranha ˜o Federal University Hospital. Diagnosis
was confirmed by identification of Leishmania sp. in Giemsa-stained
smears of bone marrow aspirates (parasitological test). The study
was conducted from August 2000 to July 2002 and information on
the individuals has been previously reported [4,7].
Sera were stored at 220uC without thawing. All patients
received adequate treatment. DTH skin reactivity assays (Mon-
tenegro test) were performed with SLA prepared as described
Parasites in logarithmic growth phase were washed twice with
PBS, lysed with Laemmli’s Buffer , sonicated, heated at 95uC
for 5 min and centrifuged for 15 min at 12,0006g at 4uC. Extracts
from 106parasites were loaded by line into an acrylamide gel. For
2D electrophoresis, parasites were lysed at 4uC with 200 mL of
Buffer A (0.5% Nonidet P40, 0.1 mM PMSF, 1 mM DTT,
10 mM Tris-HCl, pH 7.4) followed by addition of 200 mL of
phenol and vortex. The samples were centrifuged at 6,0006g for
5 min, and the aqueous phase was discarded. The proteins were
precipitated by adding 1 mL of 0.1 M ammonium acetate in
absolute methanol and centrifuged at 6,0006g for 10 min at 4uC.
The resultant pellet was washed with 80% acetone and dried. The
proteins were solubilized for 3 h at 30uC in RP3 Buffer (7 M urea,
2 M thiourea, 4% CHAPS, 40 mM Tris-HCl pH 8.8, 0.5%
ampholytes, pH 4–7), followed by 15 min of centrifugation at
12,0006g at 4uC. The amount of proteins in the supernatants was
quantified using the Quick Start Bradford Protein Assay (BioRad.
USA), and the proteins were then stocked at 20uC.
2D gel electrophoresis
Samples containing 250 mg L. chagasi protein extract in 200 mL
RP3 Buffer supplemented with DTT (50 mM) were applied by
rehydration to 11 cm IPG strips (pH 4–7). Isoelectric focusing
(IEF) was performed using a Multiphor II electrophoresis unit (GE
Healthcare, UK) at 3,500 V for 15,000 Vh. Subsequently, the
IPG strips were reduced (130 mM DTT) and alkylated (135 mM
iodoacetamide) for 15 min in equilibration buffer (0.375 M Tris-
HCl, pH 8.8, 6 M urea, 20% vol/vol glycerol, 2% wt/vol SDS).
The second dimension was run on home-casted SDS-PAGE gels
(10% or 8% wt/vol polyacrylamide) at 50 V for 30 min and then
at 160 V until the dye front reached the bottom of the gel. With
the separation in the second dimension, the proteins were
visualized by staining with PlusOneTMSilver Staining Kit or
Colloidal Coomassie staining (GE Healthcare, UK).
The electrophoresed proteins were transferred to nitrocellulose
membranes (GE Healthcare, UK) and were stained with Ponceau
S. Membranes were blocked with 5% non-fat dried milk powder in
wash solution (PBS and 0.05% Tween 20). The membranes were
probed with sera (1:1000 or 1:500), and an anti-human-IgG
Phosphatase Alkaline (PA) immunoconjugate (Sigma-Aldrich.
Germany) was used as a secondary antibody (1:2000). To measure
the recognition of sera by IgG subclasses after incubation, mouse
anti-human-IgG1, IgG2, IgG3 and IgG4 were employed. After
three washes with wash solution, an anti-mouse-IgG PA
immunoconjugate was used (1:2000). Specific IgG-PA binding
was measured with Western BlueH Stabilized Substrate for
Alkaline Phosphatase (Promega, USA).
Matching antigens and protein spots
To match antigen spots in Western blots with the corresponding
protein spot in the Coomassie gels, the blot coordinates were
defined after the Ponceau S staining pattern of the blot filter was
aligned with the spot pattern of the Coomassie gel. Only perfect
overlap (position and form) between blot-spot and Ponceau S
staining-spot was accepted (mapped spot). Spots in Western blots
without overlap with Ponceau S or Coomassie spot were defined as
The protocol that was used for in-gel digestion was based on
that in a previous publication . Briefly, gel pieces were
distained twice for 30 min at 25uC with 25 mM ammonium
bicarbonate/50% (w/v) acetonitrile, dehydrated in acetonitrile,
One of the most striking features of infection by
Leishmania chagasi is that infection leads to a spectrum
of clinical outcomes ranging from asymptomatic infection
to active disease. The existence of asymptomatic infected
people has served as an incentive to believe that an
effective vaccine is possible, but unfortunately no suc-
cessful immunological characterization of such cases was
obtained. Patients recovered from visceral leishmaniasis
show a similar immunological profile to asymptomatic
infected individuals and both exhibit a strong cell-
mediated immune response against Leishmania antigens
and are resistant to disease. Since the past decade several
approaches were undertaken to try to shed light on the
immunological profile associated with such ‘‘resistance’’ to
infections, notwithstanding antigenic recognition profile
associated to resistance to infection was not successfully
explored. In the present manuscript we describe a specific
IgG recognizing pattern associated with resistant individ-
uals (asymptomatic infected people and recovery patients
to visceral leishmaniasis). These data highlight the possi-
bility of using specific proteins in serological tests for the
identification of asymptomatic infected individuals.
Immunodominant Leishmania Antigens
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dried, and treated with trypsin (20 mg/mL, Promega, USA) in
25 mM ammonium bicarbonate pH 7.9 at 37uC for 16 h. Digests
were extracted from gel pieces with 50% (v/v) acetonitrile/water
and 0.1% (v/v) formic acid and subsequently combined and
vacuum-dried. The concentrated digests were mixed with 0.5 mL
of a-cyano-4-hydroxycinnamic acid matrix (10 mg/mL) in 50%
(v/v) acetonitrile/0.1% (v/v) trifluoroacetic acid and were spotted
onto a MALDI target plate.
MALDI-ToF/ToF mass spectrometry data
Mass spectrometric analysis was performed using MALDI ToF/
ToF-MS/MS (matrix-assisted laser desorption ionization time of
flight/time of flight-mass spectrometry) on a Shimadzu instrument
(model Axima Performance). MS data were acquired in the m/z
range of 700 to 4,000, with an accelerating voltage of 20 kV,
delayed extraction, a peak density of maximum 50 peaks per
200 Da, a minimal S/N ratio of 10 and a maximum peak at 60.
MS/MS data were acquired in the mass range of 60 Da to each
precursor’s mass, with a minimum S/N ratio of 10, a maximum
number of peaks set at 65 and a peak density maximum of 50
peaks per 200 Da.
LaunchPad 2.8.4 (Shimadzu Biotech) was used to submit the
combined MS and MS/MS data to the MASCOT protein search
engine version 2.2 using the National Center for Biotechnology
Information (NCBI) protein database. The search parameters
were as follows: no restrictions on protein molecular weight; one
tryptic missed cleavage allowed; peptide mass tolerance in the
searches was 0.2 Da for MS spectra and 0.8 Da for MS/MS
spectra. Carbamide-methylation due to treatment of sulfhydryl
with iodoacetamide and oxidation of methionine and tryptophan
were specified in MASCOT as fixed and variable modifications,
Cloning and recombinant protein purification
For expression of antigens identified by MALDI ToF/ToF-
MS/MS, coding regions were amplified by polymerase chain
reaction (PCR) and were subcloned into the pQE30 expression
vector (Qiagen, Germany). The following primers were employed
for amplification: Enolase, forward: 59-CGGGATCCATGCC-
GATCCAAAAGGTTTAC-39 and reverse: 59-CCAAGCTTT-
TACGCCCAGCCGGGGTAG-39; S-adenosylmethionine syn-
thetase, forward: CGGGATCCATGTCTGTCCACAGCATCC
TC, and reverse: 59-CCCAAGCTTTTACTCGACCATCTT
CTTGG-39; Alpha tubulin, forward: 59-CGGGATCCATGCA-
CACAGACACGCACGC-39, reverse: 59-GGGGTACCCCTTC
GCTTCACTATTTTTG -39; Heat shock protein 70, forward: 59-
shock 70, mitochondrial precursor, forward: 59-CGGGATC-
CATGTTCGCTCGTCGTGTG-39, reverse: 59-GGGGTACC
TCAACTATTACCTGAGTAGG-39 and heat shock protein
83-1, forward: 59-CGGGATCCATGACGGAGACGTTCGCG
CATGC-39. Underlined sequences in primers indicate restriction
sites for cloning.
Recombinant antigens were over-expressed in E. coli cultures,
transformed with serial pQE30s by the addition of 2 mM
isopropyl b-D-1-thiogalactopyranoside (IPTG), followed by 3 h
at 37uC incubation. Non-native bacterial lysates were subjected to
Ni-nitrilotriacetic acid agarose columns chromatography (Qiagen,
Germany). Purification was performed according to the manufac-
Soluble Leishmania antigen (SLA) from L. chagasi and recombi-
nant purified proteins were diluted in PBS buffer to 1 mg/100 mL,
and then 100 mL of each sample were placed into wells of 96-well
microtiter plates (Probind; Falcon, Becton Dickinson, USA) and
were incubated overnight at 4uC. Wash solution (PBS 16 with
0.5% Tween 20) was used three times for 10 min at room
temperature. To block wells, a blocking solution (PBS plus 0.5%
Tween 20 and 5% non-fat milk) was used for 1 h at room
temperature. Serum samples diluted at 1:100 in blocking solution
were added at 100 mL/well, and plates were then incubated for
2 h at room temperature. A new round of washes was performed
as indicated previously, followed by an incubation of 1 h at room
temperature with a 1:2000 dilution of alkaline phosphatase-
conjugated anti-human IgG antibody (Sigma-Aldrich, Germany).
Antibody excess was removed by four rounds of 10 min washes
using wash solution. The plates were developed using a
chromogenic solution of p-nitrophenylphosphate in sodium
carbonate buffer (pH 9.6) with 1 mg/mL MgCl2. The absorbance
was recorded at 405 nm.
IgG reaction with total L. chagasi protein in patients
during DTH conversion
After treatment, VL patients develop anti-Leishmania CMI, as
evidenced by positive Montenegro reaction followed by decreases
in titers of IgG against Leishmania total protein . However, even
a year after treatment and curing disease, anti-Leishmania
antibodies are still present in the sera . To understand if this
reduction in IgG recognition is associated with changes in antigen
specificity, we screened sera from patients before and after DTH
conversion for reactivity with L. chagasi total protein by Western
blot. A decrease in the number of proteins with reactivity was
observed in sera from VL patients after their recovery, and new
(Figure 1A). Next, we compared the reactivity pattern of sera
from symptomatic VL patients (DTH2), recovered patients
(DTH+), asymptomatic donors (DTH+) and uninfected volunteers
from the same endemic area (Figure 1B). Sera from post-treatment
and asymptomatic patients both of which were DTH+, showed
weak or no reactivity to a majority of the bands. A high mass
proteins group (approximately 110–130 kDa) was detected in all
naturally resistant patient samples and in 60% of post-treatment
DTH+ sera tested (Figure 1; Supplementary Figure S1; Baseline
characteristics of the study population used in Table S1). To
determine if any IgG subclass was involved in the differential
reactivity of sera between groups, IgG1, IgG2, IgG3 and IgG4
binding was tested. Only IgG1-antibody showed significant
reactivity that was similar to that of the total IgG pattern in all
cases (Table S1).
Characterization of 2D IgG recognizing by sera of VL and
To characterize the reactivity patterns associated with protec-
tion against VL, 200 mg of total L. chagasi protein was 2D
electrophoresed, electrotransferred to nitrocellulose membranes
and tested against serum samples. After the second dimension
(using 10% or 8% SDS-PAGE gel), approximately 250 spots were
obtained that had high resolution and reproducibility (Figure S2).
Membranes were tested using a pool composed of the five more
reactive sera from each group, and IgG interactions were detected.
Strikingly, we observed significant differences between groups in
the 2D Western blot (Figure 2). Results were summarized in
Immunodominant Leishmania Antigens
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Table 1. In VL, post-treatment VL and asymptomatic patients, 58,
62 and 33 spots were detected respectively (Table 1). It is
remarkable that the ratio between mapped spots versus total spots
reactions were higher when we used sera from asymptomatic (14/
33) than those found we used sera from VL (15/58) or Post-VL
(17/62) groups. Each group showed specific mapped spots,
whereas two mapped spots (202 and 204) were recognized by all
groups. In asymptomatic individuals, spots mapped were not
detected in the 110–130 kDa range, but a specific non-mapped
signal was detected (delineated in a quadrant in Figure 2). This
signal was also recognized by sera from a post-treatment VL
sample; however it was not observed when we used VL patient
MALDI ToF/ToF-MS/MS analysis of the 24 identified spots
(of which 6 spots were specifically recognized by VL patient sera, 8
spots by post-treatment VL sera, 8 spots by asymptomatic patient
sera and 2 spots recognized by all groups) resulted in the
identification of 14 (58.3%) proteins. These results, combined with
MASCOT protein identification, are summarized in Table 2.
We identified the following two proteins that specifically reacted
with sera from VL patients (DTH2): Mitochondrial 70 kDa heat
shock protein (MPT70, spot 205A) and paraflagellar rod protein 1
(PFR1, spot 207B). Additionally, eleven proteins specifically reacting
with sera from DTH+ patients were identified. Five proteins reacting
with sera from post-VL treatment patients (translation elongation
factor 1-beta [LieEF1B, spot 55], eukaryotic initiation factor 4A
[LieIF4A, spot 141A], enolase [spot 149], S-adenosylmethionine
synthetase [MAT2, spot 149A] and adaptor gamma-1 chain [spot
189]) and 6 proteins with specific reactivity with sera from
asymptomatic infected patients (disulfate isomerase [PDI, spot
148A], alpha- and beta-tubulin [spots 151A and 151B, respectively],
vacuolar ATP synthase subunit B [spot 157] and 83 kDa heat shock
protein [HSP83, spot 210]) were identified. Finally, the two proteins
recognized by all groups (spots 202 and 204) were both identified as
70 kDa heat shock protein (HSP70). The putative functions and
immunological properties of the identified proteins were retrieved
from published literature and also from the L. infantum Genome
Project database (www.genedb.org).
Figure 1. IgG reaction pattern associated to DTH conversion. Total protein extract from L. chagasi was SDS-PAGE resolved (10%
polyacrilamide) and electrotransferred to nitrocellulose filter. In all blots, sera were used at 1:1000, and secondary anti-human IgG was used at 1:2000.
A) Sera from six VL patients before and after DTH+ conversion. B) Western blot results using sera from active VL and recovered VL patients,
asymptomatic infected and non-infected individuals. Molecular weight markers are shown on the left panel.
Immunodominant Leishmania Antigens
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Characterizing the humoral response against
recombinant proteins from L. chagasi identified by
To analyze whether the identified proteins were indeed reactive
with sera from the respective groups and could be used in a
serological test for potentially asymptomatic L. chagasi infected
individuals, we measured reactivity of the following recombinant
proteins (Figure S3 for supporting information) by ELISA: alpha-
tubulin, enolase, MAT2, and HSP83, which were reactive with
DTH+ individuals sera; MPT70, which reacted with VL patients’
sera and HSP70, which reacted with sera from both groups
(Figure 3). Recombinants antigens reacted with some of the serum
samples from VL (DTH2), post-treatment (DTH+) and asymp-
tomatic patients (DTH+) (Figures 3 A–C). However, when we
normalized the titers of IgG reactive with antigens from SLA, we
observed that asymptomatic patients presented higher specific
reactivity with DTH+ antigens (Figure 3D–F). We then used a
Figure 2. 2D Western blot using sera from VL (DTH2 2), post-VL
(DTH+ +) and naturally resistant individuals (DTH+ +). Total protein
extract from L. chagasi were added to 11 cm strips (pH 4–7), followed
by SDS-PAGE (8% polyacrylamide). After Ponceau staining, the filters
were incubated with a mixture of sera at 1:1000 from VL (A) and 1:500
from post-VL (B) and naturally resistant donors (C). Only mapped spots
are indicated. Numbers corresponding with spots from 2D maps
(supplementary Figure S2). A specific DTH+ signal (none mapped) is
shown in the designated box. MW, Molecular weight markers.
Table 1. Summary of 2D Western blot results.
Total Spot Reactions5862 33
Specific Mapped Spots219189 210
114 104 148A
Non-Specific Mapped Spots 202202 202
Total Mapped Spots151814
Total Unmapped Spots43 44 19
Reproducible spots reacting with each group are indicated. Mapped and non-
mapped spots are defined in Materials and Methods. Asterisks indicate low
Immunodominant Leishmania Antigens
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Table 2. Summary of MALDI ToF/ToF-MS/MS results.
MS/MS peptide (Ion Score)
HSP 70-like mt
Paraflagellar rod protein 1
Translation elongation factor 1-beta
Eukaryotic initiation factor 4A
Adaptor gamma-1 chain
Disulfate isomerase PDI
Tubulin, beta chain
R.LHFFMMGFAPLTSR.G (5)R.YLTASALFR.G (71)
Tubulin, alpha chain
Vacuolar ATP synthase subunit B
HSP 70-related protein
70 kDa heat shock protein
R.LVTFFTEEFKR.K (72)R.LVTFFTEEFKR.K (44)R.ARFEELCGDLFR.S (52)K.SQIFSTYADNQPGVHIQVFEGER.A (72)
Note. References are related to the significance of these antigens with regard to drug resistance, host parasite survival or immunological properties.
*Mascot Score, protein overall scores greater than 20 are significant (P,0,05). MW, molecular weight; Ip, isoelectric point.
Immunodominant Leishmania Antigens
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combination of recombinants proteins composed of enolase,
MAT2, alpha-tubulin and HSP83 (MIX) to test the sensitivity in
the sera from asymptomatic patients and their specificity against
sera from patients with Chagas disease, which presents high
cross-reactivity with Leishmania antigens . We verified that
asymptomatic individuals had a higher reaction with MIX than
SLA, and sera from patients with Chagas disease showed lower
cross-reactivity (Figure 4).
Figure 3. ELISA with individual recombinant proteins. SLA and obtained recombinants proteins (10 mg/mL) were tested for reactivity with sera
(diluted 1:100) from those groups shown above the panels. Absolute OD obtained (A–C) and the ratio between antigen-OD versus SLA-OD for each
serum sample (D–F)) are shown. The dotted line shows the OD ratio at 1. Medians are shown as horizontal lines.
Figure 4. Mixture of DTH+ + antigens in serological diagnosis of asymptomatic infected individuals. ELISA results were obtained using
10 mg/mL SLA or DTH+ recombinant antigen mixture (MIX, contained 2.5 mg/mL of each of the following proteins: enolase, MAT2, alpha tubulin and
HSP83). Sera from asymptomatic L. chagasi infected patients, Chagas-infected patients were used (1:100).
Immunodominant Leishmania Antigens
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The decrease in anti-Leishmania IgG titers during conversion to
DTH+ in effectively treated VL patients has been previously
associated with the development of cell-mediated immune
responses . Herein, we show that decreased antibody levels
in VL-related states and positive DTH reactions are associated
with a reduction in the reactivity against most of the parasite
proteins and, more relevant, the recognition of previously
unrecognized proteins. These observations indicate that impor-
tant changes in the humoral response occurs during DTH
conversion and reveals a reactivity pattern associated with
recovery and natural resistance to VL that is characterized by
high-mass group proteins (approximately 110–130 kDa). These
high-mass proteins did not react with sera from VL patients,
indicating that some specific immunodominant antigens are
specifically associated with DTH conversion and putatively with
protection against VL. Unfortunately, high-mass group proteins
were not 2D-resolved, and their identification was not possible;
however, 2D-recognizing map analyses of sera from VL patients
and DTH+ individuals (post-VL or naturally resistant) showed
other immunodominant antigens whose recognition was DTH
status-dependent. A lower number of reactive spots were
observed in sera from DTH+ individuals living in the endemic
area who are presumably naturally resistant. It is noteworthy that
sera from post-VL patients (DTH+) reacted with a larger number
of proteins, which suggests that post-VL patients likely share
antigenic recognition patterns with both VL patients and
naturally resistant individuals which reinforces the idea that
some antigens are specifically associated with positive DTH
response and therefore protection against VL.
Proteomic analysis revealed the identification of 14 antigens,
including proteins not previously reported as antigenic, such as
adaptor gamma-1 chain, MPT70 and PFR1. Among the
identified antigens associated with the post-VL subset, four of
them (LieIF4A, LieEF1B, enolase and MAT2) have been
reported as highly expressed in drug-resistant strains [12–14]
and have been implicated in the survival of the parasite inside the
host [15,16]. For the six identified antigens that reacted with sera
from the VL naturally resistant people (PDI, alpha- and beta-
tubulin, vacuolar ATP synthase subunit B and HSP83), drug
resistance implications and host parasite survival properties have
been previously described too [13,17–21]. The stress state
imposed on the parasite in a DTH+ environment (resulting
either from drug treatment or natural resistance) can induce the
overexpression of these proteins, and this fact may explain their
immunodominant antigenicity in these individuals. It is notable
that LieIF4A protein was described as antigen reactive with sera
from VL patients infected by L. donovani from India  and we
found a clear reactivity against this protein using sera from post-
VL patients. It could be interpreted as yet another indication that
IgG pattern recognition in post-VL patients preserved reactivity
against some antigens of active illness. In addition, it was already
observed that a Th1 activation response, associated with healing,
was induced in cutaneous leishmaniasis patients’ PBMC by
recombinant LieIF4A . We consider the hypothesis that some
correlation may exist between the antigenicity of DTH+
recognized proteins and its capacity to induce anti-Leishmania
cellular responses. On the other side, it is possible that the highly
immunogenic proteins, such as HSP70 [22–24], are implicated in
its recognition by all groups.
To determine if the immunodominant antigens identified could
serve as serological markers, some of them were expressed as
recombinant proteins, purified and then employed as antigen in
ELISA (Figure 3). When we normalized to SLA, enolase exhibited
a remarkable increase in reactivity against sera from post-VL
group respect to other antigens. Moreover, an increased enolase
recognition was observed using sera from asymptomatic people,
strengthening the idea that antigens recognized by post-VL sera
could also be reactive in VL patients and resistant people sera. In
the other hand, alpha tubulin and HSP83, showed a remark
recognition increase from asymptomatic donors’ sera. Meanwhile,
MTP70 that was identified as a VL-specific protein did not react
with any groups. There are four not identical copies of MPT70 in
L. infantum genome (www.genedb.org), and only one of them was
cloned to obtain MPT70 recombinant protein. Future studies with
the other 3 variants will shed light about the real serological
properties of these proteins. Finally, and as expected, HSP70
reacted with several serum samples from all groups.
Lastly, we proved that a mixture of DTH+ antigens (enolase,
MAT2, alpha-tubulin and HSP83) had higher reactivity with sera
from asymptomatic individuals than SLA and lower cross-
reactivity with sera from patients with Chagas disease. Our results
are the first description of several antigens that are immunodo-
minant in DTH+ individuals (post-VL and naturally resistant
people). Future studies using these antigens may help to identify
potent serological tools that could be useful for determining patient
disease status as well as new anti-Leishmania vaccine candidates.
associated to DTH conversion. Total protein extract from
L. chagasi was SDS-PAGE resolved (10% polyacrilamide) and
electrotransferred to nitrocellulose filter. In all blots, sera were
used at 1:1000 (1:100 in B - line 5) and secondary anti-human IgG
(or subclasses IgG1, IgG2, IgG3 and IgG4 in E and F) was used at
1:2000. A) Sera from VL patients. B) Sera from uninfected people.
C) Sera from post-VL patients (DTH+). D) Sera from asymptom-
atic infected people. E) VL sera mix (VL0678, VL0669, VL0660,
VL0671, VL0666). F) Asymptomatic sera mix (576, 421, 689, 011,
417). Under panels are show the serum code. Asterisk over panel
indicate filters with DTH+ patter (110–130 KDa proteins). MW,
Molecular weight markers. Baseline characteristics of the study
population are shown in supplementary Table S1.
Characterization of IgG reaction pattern
200 mg of total protein from L. chagasi was 2D resolved on 11 cm
strips with a pH range of 4–7 (first dimension) and SDS-PAGE.
The numbers of mapped spots are indicated. A) Gel after
electrotransfer to a nitrocellulose filter and Ponceau Red staining
(8% polyacrylamide in second dimension). B) Gel with 10%
polyacrylamide and Colloidal Coomassie staining. C) Gel with
10% polyacrylamide and Silver staining.
2D proteomic map of L. chagasi. Approximately
identified by proteomic approaches. Identified E. coli (M15)
overexpressing recombinant antigens (fused to a 6xHis tag) were
used for Ni-affinity chromatography. Obtained purified proteins
resolved in 10% polyacrylamide gel stained with Comassie are
shown. MW, Molecular weight marker.
Purified recombinant antigens of L. chagasi
used in this study. Age, sex, serum code, OD value obtained
from Elisa test, and DTH status for each of the groups are show.
Baseline characteristics of the sera donors
Immunodominant Leishmania Antigens
www.plosntds.org8 June 2012 | Volume 6 | Issue 6 | e1687
The ´o de Arau ´jo-Santos provided assistance with statistical analyses and
Conceived and designed the experiments: DRA MS AB MBN. Performed
the experiments: DRA LVA ESA JRASP DA AJC. Analyzed the data:
DRA MSP AB MBN. Contributed reagents/materials/analysis tools: MSP
DA AJC. Wrote the paper: DRA LVA MBN.
1. Wilson ME, Jeronimo SM, Pearson RD (2005) Immunopathogenesis of infection
with the visceralizing Leishmania species. Microb Pathog 38: 147–
2. Ribeiro-de-Jesus A, Almeida RP, Lessa H, Bacellar O, Carvalho EM (1998)
Cytokine profile and pathology in human leishmaniasis. Braz J Med Biol Res 31:
3. Oliveira JM, Fernandes AC, Dorval ME, Alves TP, Fernandes TD, et al. (2010)
[Mortality due to visceral leishmaniasis: clinical and laboratory characteristics].
Rev Soc Bras Med Trop 43: 188–193.
4. Caldas A, Favali C, Aquino D, Vinhas V, van Weyenbergh J, et al. (2005)
Balance of IL-10 and interferon-gamma plasma levels in human visceral
leishmaniasis: implications in the pathogenesis. BMC Infect Dis 5: 113.
5. Jeronimo SM, Teixeira MJ, Sousa A, Thielking P, Pearson RD, et al. (2000)
Natural history of Leishmania (Leishmania) chagasi infection in Northeastern
Brazil: long-term follow-up. Clin Infect Dis 30: 608–609.
6. Davies CR, Kaye P, Croft SL, Sundar S (2003) Leishmaniasis: new approaches
to disease control. BMJ 326: 377–382.
7. Aquino DMC, Caldas AJM, Miranda JC, Silva AAM, Barral-Netto M, et al.
(2010) Epidemiological study of the association between anti-Lutzomyia
longipalpis saliva antibodies and development of delayedtype hypersensitivity
to Leishmania antigen. Am J Trop Med Hyg 83: 825–827.
8. Reed SG, Badaro ´ R, Masur H, Carvalho EM, Lorenco R, et al. (1986) Selection
of a skin test antigen for American visceral leishmaniasis. Am J Trop Med Hyg
9. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the
head of bacteriophage T4. Nature 227: 680–685.
10. Shevchenko A, Wilm M, Vorm O, Mann M (1996) Mass spectrometric
sequencing of proteins silver-stained polyacrylamide gels. Anal Chem 68: 850–
11. Vexenat Ade C, Santana JM, Teixeira AR (1996) Cross-reactivity of antibodies
in human infections by the kinetoplastid protozoa Trypanosoma cruzi,
Leishmania chagasi and Leishmania (viannia) braziliensis. Rev Inst Med Trop
Sao Paulo 38: 177–185.
12. Singh G, Chavan HD, Dey CS (2008) Proteomic analysis of miltefosine-resistant
Leishmania reveals the possible involvement of eukaryotic initiation factor 4A
(eIF4A). Int J Antimicrob Agents 31: 584–586.
13. Kumar A, Sisodia B, Misra P, Sundar S, Shasany AK, et al. (2010) Proteome
mapping of overexpressed membrane-enriched and cytosolic proteins in sodium
antimony gluconate (SAG) resistant clinical isolate of Leishmania donovani.
Br J Clin Pharmacol 70: 609–617.
14. Drummelsmith J, Girard I, Trudel N, Ouellette M (2004) Differential protein
expression analysis of Leishmania major reveals novel roles for methionine
adenosyltransferase and S-adenosylmethionine in methotrexate resistance. J Biol
Chem 279: 33273–33280.
15. Vickers TJ, Wyllie S, Fairlamb AH (2004) Leishmania major elongation factor
1B complex has trypanothione S-transferase and peroxidase activity. J Biol
Chem 279: 49003–49009.
16. Vanegas G, Quinones W, Carrasco-Lopez C, Concepcion JL, Albericio F, et al.
(2007) Enolase as a plasminogen binding protein in Leishmania mexicana.
Parasitol Res 101: 1511–1516.
17. Vergnes B, Gourbal B, Girard I, Sundar S, Drummelsmith J, et al. (2007) A
proteomics screen implicates HSP83 and a small kinetoplastid calpain-related
protein in drug resistance in Leishmania donovani clinical field isolates by
modulating drug-induced programmed cell death. Mol Cell Proteomics 6: 88–
18. Prasad V, Kumar SS, Dey CS (2000) Resistance to arsenite modulates levels of
alpha-tubulin and sensitivity to paclitaxel in Leishmania donovani. Parasitol Res
19. Mojtahedi Z, Clos J, Kamali-Sarvestani E (2008) Leishmania major:
identification of developmentally regulated proteins in procyclic and metacyclic
promastigotes. Exp Parasitol 119: 422–429.
20. Ben Achour Y, Chenik M, Louzir H, Dellagi K (2002) Identification of a
disulfide isomerase protein of Leishmania major as a putative virulence factor.
Infect Immun 70: 3576–3585.
21. Bakker-Grunwald T (1992) Ion transport in parasitic protozoa. J Exp Biol 172:
22. Skeiky YA, Benson DR, Guderian JA, Whittle JA, Bacelar O, et al. (1995)
Immune responses of leishmaniasis patients to heat shock proteins of Leishmania
species and humans. Infect Immun 63: 4105–4114.
23. Rafati S, Gholami E, Hassani N, Ghaemimanesh F, Taslimi Y, et al. (2007)
Leishmania major heat shock protein 70 (HSP70) is not protective in murine
models of cutaneous leishmaniasis and stimulates strong humoral responses in
cutaneous and visceral leishmaniasis patients. Vaccine 25: 4159–4169.
24. Quijada L, Requena JM, Soto M, Alonso C (1998) Analysis of the antigenic
properties of the L. infantum Hsp70: design of synthetic peptides for specific
serodiagnosis of human leishmaniasis. Immunol Lett 63: 169–174.
25. Forgber M, Basu R, Roychoudhury K, Theinert S, Roy S, et al. (2006) Mapping
the antigenicity of the parasites in Leishmania donovani infection by proteome
serology. PLoS One 1: e40.
26. Skeiky YA, Guderian JA, Benson DR, Bacelar O, Carvalho EM, et al. (1995) A
recombinant Leishmania antigen that stimulates human peripheral blood
mononuclear cells to express a Th1-type cytokine profile and to produce
interleukin 12. J Exp Med 181:1527–37.
27. Probst P, Skeiky YA, Steeves M, Gervassi A, Grabstein KH, et al. (1997) A
Leishmania protein that modulates interleukin (IL)-12, IL-10 and tumor necrosis
factor-alpha production and expression of B7-1 in human monocyte-derived
antigen-presenting cells. Eur J Immunol 27: 2634–2642.
28. Gupta SK, Sisodia BS, Sinha S, Hajela K, Naik S, et al. (2007) Proteomic
approach for identification and characterization of novel immunostimulatory
proteins from soluble antigens of Leishmania donovani promastigotes.
Proteomics 7: 816–823.
29. Kumari S, Samant M, Misra P, Khare P, Sisodia B, et al. (2008) Th1-
stimulatory polyproteins of soluble Leishmania donovani promastigotes ranging
from 89.9 to 97.1 kDa offers long-lasting protection against experimental
visceral leishmaniasis. Vaccine 26: 5700–5711.
30. Bhowmick S, Ali N (2009) Identification of novel Leishmania donovani antigens
that help define correlates of vaccine-mediated protection in visceral
leishmaniasis. PLoS One 4: e5820.
31. Rico AI, Girones N, Fresno M, Alonso C, Requena JM (2002) The heat shock
proteins, Hsp70 and Hsp83, of Leishmania infantum are mitogens for mouse B
cells. Cell Stress Chaperones 7: 339–346.
32. Echeverria P, Dran G, Pereda G, Rico AI, Requena JM, et al. (2001) Analysis of
the adjuvant effect of recombinant Leishmania infantum Hsp83 protein as a tool
for vaccination. Immunol Lett 76: 107–110.
33. Rasouli M, Zavaran Hoseini A, Kazemi B, Alborzi A, Kiany S (2009) Expression
of recombinant heat-shock protein 70 of MCAN/IR/96/LON-49, a tool for
diagnosis and future vaccine research. Iran J Immunol 6: 75–86.
34. Quijada L, Requena JM, Soto M, Gomez LC, Guzman F, et al. (1996) Mapping
of the linear antigenic determinants of the Leishmania infantum hsp70
recognized by leishmaniasis sera. Immunol Lett 52: 73–79.
Immunodominant Leishmania Antigens
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