Skip to main content
NCBI home page
PLOS One logoLink to PLOS One
. 2016 Jan 15;11(1):e0147076. doi: 10.1371/journal.pone.0147076

Prediction of T Cell Epitopes from Leishmania major Potentially Excreted/Secreted Proteins Inducing Granzyme B Production

Ikbel Naouar 1,2, Thouraya Boussoffara 1,2,*, Mehdi Chenik 2,3, Sami Gritli 4, Melika Ben Ahmed 1,2, Nabil Belhadj Hmida 1,2, Narges Bahi-Jaber 1,5, Rafika Bardi 6, Yousr Gorgi 6, Afif Ben Salah 1,2, Hechmi Louzir 1,2
Editor: Silke Appel7
PMCID: PMC4714855  PMID: 26771180

Abstract

Leishmania-specific cytotoxic T cell response is part of the acquired immune response developed against the parasite and contributes to resistance to reinfection. Herein, we have used an immune-informatic approach for the identification, among Leishmania major potentially excreted/secreted proteins previously described, those generating peptides that could be targeted by the cytotoxic immune response. Seventy-eight nonameric peptides that are predicted to be loaded by HLA-A*0201 molecule were generated and their binding capacity to HLA-A2 was evaluated. These peptides were grouped into 20 pools and their immunogenicity was evaluated by in vitro stimulation of peripheral blood mononuclear cells from HLA-A2+-immune individuals with a history of zoonotic cutaneous leishmaniasis. Six peptides were identified according to their ability to elicit production of granzyme B. Furthermore, among these peptides 3 showed highest affinity to HLA-A*0201, one derived from an elongation factor 1-alpha and two from an unknown protein. These proteins could constitute potential vaccine candidates against leishmaniasis.

Introduction

Leishmaniasis represents a heterogeneous group of diseases with an estimated incidence of 2 million cases annually worldwide [1]. They are caused by protozoan parasites of the genus Leishmania and are transmitted by the bite of infected sand flies. The disease is characterized by a spectrum of clinical manifestations determined by the species of Leishmania and the immune response of the host to the parasite [2]. It ranges from asymptomatic infections to cutaneous or fatal visceral forms. Most individuals who developed leishmaniasis or symptomless infection are resistant to subsequent infections, which makes vaccine development rational [3]. Studies of anti-Leishmania vaccine candidates have advanced in recent years due to the understanding of the cell-mediated immunological mechanisms for controlling infection. However, no efficient vaccine is available for human use as of today and Leishmania vaccine development has proven to be a difficult and challenging task.

In common with other intracellular pathogens, cellular immune responses are critical for protection against leishmaniasis [4]. Considerable evidence suggests that Leishmania major infection induces the development of a Th1 response that not only controls the primary infection but also results in a lifelong immunity to reinfection. Protection against Leishmania infection has been shown to involve CD4+ and CD8+ T cells [5–9]. Indeed, peripheral blood mononuclear cells (PBMCs) obtained from individuals with active or healed localized cutaneous leishmaniasis proliferate and produce Th1 type cytokines, when stimulated in vitro with Leishmania antigens [10–12]. However, previous reports indicate the implication of CD8+ T cells in immunoprotective mechanisms in CL as well as the establishment of a Th1 response, mainly through the production of IFN-γ [12] Although cytokine production is thoroughly analyzed, the involvement of cytotoxic activity in protection remains undefined.

Previously, we have shown that cytotoxic activity specific of Leishmania major (L. major) is developed by individuals living in areas of L. major transmission [13] and seems to play a crucial role in resistance to re-infection (Louzir H, 2005, unpublished data). Similar data suggest that CD8+ T cells may have a protective role in subclinical infection [14]. Contrastingly, evidence has been accumulated regarding the role of CD8+ T cells in the pathophysiology of CL. Indeed, these cells have been involved in the chronicity of Leishmania infection by exacerbating the tissue lesions, as described in mucocutaneous leishmaniasis caused by L. braziliensis [14–16]. Such controversy regarding the role of cytotoxicity in the pathogenesis of human leishmaniasis indicates that the functions of CD8+ T cells remain to be established. Furthermore, conflicting data about the route of activation of CD8+ T cells in leishmaniasis exist, since Leishmania resides within the parasitophorous vacuole of the macrophage and it is not clear how these cells present Leishmania antigens to CD8+ T cells through class I MHC [17–19]. Several data suggest that external or secreted Leishmania antigens are able to reach macrophage cytosol to be presented by class I HLA molecules, which is a prerequisite for CD8+ T cell activation [17–19].

Previously, we also have characterized a set of 33 Leishmania proteins that are potentially secreted by the parasite in the phagolysosomal vacuole [20].

Herein, we have first used immuno-informatic tools to select nonameric peptides derived from the 33 Leishmania major excreted/secreted (LmES) proteins previously described based on the binding motifs of the class I MHC: HLA-A*0201, which is the most frequent HLA allele in the Tunisian population (32.5%) [21]. Potentially ES proteins have been reported to contain antigens highly immunogenic and protective in vaccine models [17, 22–25]. Evidence has been shown regarding the immunogenicity of Leishmania ES proteins recovered from human cutaneous leishmaniasis [26]. In silico peptide prediction was followed by experimental validation of the capacity of these peptides to bind to HLA-A2 and the analysis of their immunogenicity in naturally-exposed individuals.

Materials and Methods

Selection of study subjects

Peripheral blood was obtained from 6 HLA-A*0201 positive and 6 HLA-A*0201 negative donors recovered from zoonotic cutaneous leishmaniasis (ZCL) living in an area of high transmission of L. major parasite (Central Tunisia). These individuals were selected based on (i) clinical criteria showing the presence of ZCL scars, (ii) positivity of the leishmanin skin test (LST) reactivity, and/or (iii) positive lymphoproliferative response to soluble Leishmania antigens (SLA) [immune individuals]. Screening of HLA-A*0201 positive individuals was done using a lymphocytotoxicity test. HLA subtype A*0201 was confirmed by PCR using HLA SSP ABC Typing Kit (One Lambda Inc., Canoga Park, CA). HLA-A*0201 positive healthy individuals living outside endemic areas without any lymphoproliferative response to SLA were included as control groups. The main clinical and laboratory features of the selected individuals are described in Table 1. This study has obtained the Ethical Committee approval of the Pasteur Institute of Tunis (protocol number 07–0018). Individuals were included in the study after providing informed written consent.

Table 1. Clinical and laboratory main features of the study subjects.

Sex Age(years) LCZ scars(Y/N) LST (mm) Proliferaion (SLA) SI HLA-A Typing
ZCL04 F 58 Y 8,5 41.87 A2/01 A24
ZCL05 F 50 Y 7 63.22 A2/01 A24
ZCL23 F 40 Y ND 20.42 A2/01 A1
ZCL25 M 41 N 7 44.62 A2/01 A26
ZCL29 M 32 N 10 45.4 A2/01 A30
ZCL34 M 46 N ND 35.68 A2/01 A30
ZCL01 M 39 Y 12 109 A1 A28
ZCL03 F 42 Y 7.5 79 A11 A32
ZCL07 F 41 Y 8.5 14 A26 A30
ZCL14 F 24 Y 14 19 A1 A23
ZCL22 F 20 N ND 12.5 A24 A11
ZCL24 F 24 Y ND 6.8 A3 A26
T1 F 50 N ND 1.6 A2/01 A25/01
T2 F 42 N ND 2.02 A2/01 A30/01
Open in a new tab

ND: Not determined; SI: Stimulation Index; SLA: Soluble Leishmania Antigens

LST: Leishmanin Skin Test; F: Female; M: Male; Y/N: Yes/No.

Epitope prediction and peptide synthesis

A set of 33 L. major genes encoding proteins that are potentially ES proteins by the parasite have previously been described in our laboratory [20].

All protein sequences were submitted to analysis by computerized HLA-binding prediction based on the freely accessible online databases: “Syfpeithi”: http://www.syfpeithi.de/bin/MHCServer.dll/EpitopePrediction.htm, HLA-peptide binding prediction site supplied by: “BIMAS”: http://www-bimas.cit.nih.gov/molbio/hla_bind, “RANKPEP”: http://www.http://bio.dfci.harvard.edu/MIF/RANKPEP, and “NetMHC”: http://www.cbs.dtu.dk/services/NetMHC. “Syfpeithi”, “BIMAS”, and “NetMHC” programs provide peptide sequences that are likely to be presented by the HLA-A*0201 molecules.

The probability for the peptides to be cleaved in the proteasome was predicted by “RANKPEP” along with a ranking or score. All peptides predicted with at least 3 softwares were selected and purchased from Intavis Bioanalytical Instruments (Cologne, Germany). Stock solutions of single peptides (20mg/mL) were produced by dissolving freeze-dried peptides in DMSO (Sigma-Aldrich, St. Louis, MO) and kept at -80°C until use.

Parasites

L. major (Zymodeme MON25; MHOM/TN/94/GLC94) isolated from skin lesions of patients with CL was used in the present study. Parasites were cultivated on NNN medium at 26°C and then were progressively adapted to RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO) containing 2mM L-Glutamine (Sigma-Aldrich, St. Louis, MO), 100U/mL Penicillin (Sigma-Aldrich, St. Louis, MO), 100mg/mL Streptomycin (Sigma-Aldrich, St. Louis, MO), and 10% heat-inactivated fetal calf serum (Sigma-Aldrich, St. Louis, MO). Stationary-phase metacyclic promastigotes were used to infect macrophages.

Cell line

The T2 cell line is a human tumor cell line that expresses HLA-A*0201 and lacks TAP1 and TAP2 transporters [T2 (174 x CEM.T2), (ATCC® CRL-1992TM)]. It was kindly provided to us by Dr. Salem Chouaib (Gustave Roussy Institute, France).

Detection of peptides binding to HLA-A*0201 molecules on T2 cells

The affinity of peptides for HLA-A*0201 molecules was evaluated by using the stabilization assay as previously described [27]. Briefly, T2 cells were incubated with human β2-microglobulin at a final concentration of 10μg/mL in the presence or not of peptides at 10μg/mL for 16h at 37°C in 5% CO2. Cells were then incubated with 5μg/mL Brefeldin A (Sigma-Aldrich, St. Louis, MO) for 2h at 37°C. Expression of HLA-A*0201 on T2 cells was then determined by staining with fluorescein isothiocyanate-labelled anti-HLA-A2 antibody (BD Biosciences, San Jose, CA) and analyzed by flow cytometry using FACScan (BD Biosciences, San Jose, CA). Results were expressed in relative fluorescence intensity (RFI) calculated as the percentage increase of the mean fluorescence above that of the negative controls [28].

In vitro stimulation of PBMCs with peptides

To assess whether the selected peptides could stimulate or not CD8+ T cells, we have analysed the induction of GrB and IFN-γ by stimulated PBMCs from healed ZCL individuals. PBMCs separated from heparinized blood samples using Ficoll-Hypaque (Sigma-Aldrich, St. Louis, MO) density gradient centrifugation were resuspended in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO) supplemented with 2mM L-Glutamine (Sigma-Aldrich, St. Louis, MO), 1mM sodium pyruvate (Gibco, Invitrogen, Grand Island, NY), 100U/mL Penicillin (Sigma-Aldrich, St. Louis, MO), 100μg/mL Streptomycin (Sigma-Aldrich, St. Louis, MO), 10mM HEPES (Gibco, Invitrogen, Grand Island, NY), 20μg/mL Gentamicin (Gibco, Invitrogen, Grand Island, NY), 1X non-essential amino acids (Gibco, Invitrogen, Grand Island, NY), 2-mercaptoethanol (Gibco, Invitrogen, Grand Island, NY), and 10% (v/v) heat-inactivated human AB serum (Sigma-Aldrich, St. Louis, MO), [complete medium] at a concentration of 1.0x106 cells/mL. Peptide pools were prepared instantly by dilution with phosphate buffered saline and then added to the cell culture at a final concentration of 1μg/mL. In some experiments, peptides were added separately to the culture at a concentration of 20μg/mL. As positive control, PBMCs were stimulated with 10ng/mL of phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, St. Louis, MO) and 50ng/mL Ionomycin (Sigma-Aldrich, St. Louis, MO). All cultures were incubated at 37°C in 5% CO2 for 5 days. Culture supernatants were then harvested and frozen at -80°C until use.

Granzyme B, IFN-γ, and IL-10 ELISA assays

Granzyme B (GrB), IFN-γ, and IL-10 levels in culture supernatants were quantified with an enzyme-linked immunosorbent (ELISA) assay (MABTECH AB, Nacka Strand, Sweden) for the first one and OptEIA set ELISA assay kit (BD Biosciences, San Jose, CA) for the others. The results were expressed as pg/mL based on the standards provided by the kits. Quantification thresholds were fixed to 100pg/mL for GrB, 45pg/mL for IFN-γ, and 20pg/mL for IL-10.

Statistical analyses

Statistical analyses were carried out by using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA). Mann-Whitney test was used to compare the induction of GrB and IFN-γ between the different study groups. Correlation between GrB and IFN-γ levels induced by peptide pools or individual peptides were estimated by use of Spearman’s rank order correlation coefficient. A classification of the peptide pools according to their induction of GrB was achieved using Matlab 7.0 (Mathworks, Inc., Natick, MA). A Kruskal-Wallis test was performed to compare the rank of peptide pools.

Results

Selection of potential HLA-A*0201-binding peptides within LmES proteins

The sequence of 33 different clones of potentially ES proteins has been used. Based on computer software predictions, putative class I HLA-restricted T cell epitopes were identified. Twenty proteins were able to generate a total of 78 nonameric peptides that could be loaded by HLA-A*0201 molecule (Table 2). Subsequently, we have evaluated the binding affinity of these peptides to HLA-A*0201 molecules by class I HLA stabilization assay. This assay measures the increase of HLA-A*0201 molecules induced on T2 cells following exposure to exogenous HLA-A*0201 binding peptides, with high affinity peptides inducing HLA-A*0201 up-regulation more strongly than low-affinity peptides. Individual results are shown on Fig 1A. The 78 tested peptides were classified into 3 groups regarding the percentage of RFI. Six peptides namely, D6, E1, F6, G1, G2, and G3 showed the highest percentage of RFI increase (RFI > 200%), 50 peptides showed intermediate affinity (RFI ranges from 100 to 200%), and 22 peptides had a weak affinity (RFI < 100%) (Fig 1B).

Table 2. Characteristics of in silico predicted HLA-A*0201-restricted peptides for LmES proteins.

Protein peptide Start Position SEQUENCE RANKPEPa SYFPEITYb BIMASa NET MHC MATRIXa NET MHC ANNa
A1 85 ALQEETHVL 82 75% 35.9102 25.522 101
A2 427 YMAQKAEEV 74.05 69.44% 113.2229 20.067 48
Pr 9.1 (LmjF.14.0820) A3 259 KLTVSSAAV 93 - - 23.091 1580
A4 92 VLGSHVQTL 86 75% 83.5270 25.372 -
A5 182 LLRQETARL 82 72.22% - 23.016 1468
A6 362 HLMGQLNEL 79 83.33% - 25.566 274
Pr 9.2(Ribosomalprotein S18) A7 107 RLRDDLERL 70 69.44% 7.5019 20.177 1437
A8 45 YLLDVSTLL 94 6.44% 1490.7110 26.521 100
A9 198 NLIDFNFKL 72 72.22% 1930.3919 20.772 152
Pr 12 (Ubiquitin A10 185 LLKDSFAFL 85 66.66% - 22.603 653
protein ligase: LmjF.07.0280) A11 230 CLLDSFKEL 75 66.66% 615.7285 22.474 -
A12 167 VLEENRTTL 73 66.66% - 20.196 -
B1 9 VLCALLFCV 68 72.22% 1577.3003 25.092 562
B2 387 KLHPVYDKV 66 69.44% 178.9225 24.676 318
Pr 13 (LmPDI) B4 259 ALKGSLVAV 91 83.33% - 22.517 344
B5 157 EMASMITKV 88 63.88% - - 1171
B6 63 DMLAGIATL 69 80.55% - - 716
B7 321 LLSAQIARL 93 77.77% 83.5270 23.94 771
B8 256 LLFDELTAL 84 80.55% 1267.1043 24.159 174
Pr 15 (LmjF.15.0410) B9 737 RLMQCVQQL 81 69.44% 181.7940 23.118 1445
B10 33 SLVVVSASL 88 72.22% - 23.241 5167
B11 29 SLCRSLVVV 86 77.77% - 23.281 5111
B12 22 HLVAPLASL 82 80.55% - 23.516 2134
C1 395 ALNDALWAV 96 80.55% 4919.0652 27.307 45
C2 77 RLLVDLAQL 83 77.08% 181.7940 21.354 -
Pr 20.1 (Chaperonin C3 175 IVVDAIMSV 101 - 97.5615 - 465
subunit alpha: LmjF.32.3270) C4 367 VIAGTSNAV 77 69.44% - - 835
C5 128 AMREALRYL 76 72.22% - - 349
C6 213 GVFDAAISI 82 - 13.8482 - 600
Pr 20.2 (LmjF.36.2650) C7 69 QVGAFLEGL 50 55.55% 8.0051 - -
C8 145 GLDYSEELL 46 55.55% 4.1870 - -
Pr 22 (LmjF.05.0710) C9 111 RVAASVAAV 95 63.88% 13.9973 - 10621
C10 221 GTDDTVAAV 64 63.88% 3.6438 - 8253
C11 141 TIPSFIVRV 88 69.44% 83.5841 - 2164
C12 68 RLLEGSAIM 79 61.11% 30.8995 - 582
Pr 22.1 (Ribosomal D1 127 LIQQRHIAV 66.05 58.33% 16.2578 - 1987
protein S9: LmjF.36.1250) D2 134 AVAKQIVTI 95 66.66% - - 8403
D3 103 ILERRLQTI 78 69.44% - - 2278
Pr 27 (similar to LAEL147_000045800) D4 24 NMMAVVGLL 81 63.88% 17.0684 - 6564
D5 836 KLEDEVFAL 83 72.22% 261.7205 23.158 170
D6 892 ELLGNLEEV 79 75% 21.7519 24.38 535
Pr 31 (LmjF.34.0680) D7 690 RMADEVQRL 77 69.44% 145.4898 20.464 520
D8 135 RLAVSLHEL 81 80.55% 49.1335 22.162 -
D9 648 LLGPAYQSI 78 - 26.6036 - 729
D10 781 VIAEEPLYV 77 - 366.6129 - 1130
D11 40 PLSAVISPV 81 - - 25.673 641
D12 320 LLPAPLVSV 90 86.11% 271.9483 26.986 504
Pr 37 (LmjF.36.3860) E1 575 MLLWTAVAV 82 69.44% 437.4821 25.381 162
E2 135 YLRTFPAAL 70 72.22% - 27.977 -
E3 336 RLAGFLAGL 78 88.88% 186.7074 24.784 516
E4 385 CLALIAWRV 67 61.11% 521.1640 21.709 605
Pr 38 (similar to LTRL590_180019400) E5 69 VVAGMLRWV 65 63.88% 26.1750 - 1295
E6 134 PLSPATRRL 64 58.33% - 22.415 1722
E7 92 MVLNAMAWL 78 - 148.7302 - 2967
Pr 57 (Ribosomal E8 53 KIMEAITVV 105 72.22% 478.8259 - 384
protein S16: LmjF.26.0880/ LmjF.26.0890) E9 115 FLAYDKFLL 115 61.11% 569.9488 - 343
Pr 66 (LmjF.26.0880/ LmjF.26.0890) E10 461 HIFDRVAGV 78 75% - - 361
E11 75 ALNQFTKVL 79 69.44% 33.2826 22.002 2323
Pr 68 (Ribosomal E12 179 AIVKDMARL 88 61.11% 6.7559 - 7557
protein L7/ F1 136 GLQEVTRAI 83 66.66% 8.5549 - 717
L12-like protein: LmjF.07.0500) F2 153 VIANNVDPV 69 69.44% 18.3225 - 2773
F3 211 TLKNLIRSV 87 72.22% - - 735
Pr 74 (elongation F4 215 TLLDALGML 93 75% 96.8962 21.586 1262
factor proteasome F5 137 ALLAFTLGV 90 80.55% 977.9011 27.727 287
1-alpha: LmjF.17.0082) F6 142 TLGVKQMVV 78 58.33% 28.5163 - 2243
F7 263 KLVRELFRV 90 69.44% 3247.3829 25.63 506
Pr 77 (Probable F8 306 TMLELLTQL 89 75% 538.3123 20.999 342
regulatory ATPase F9 404 ALRERRMKV 80 72.22% 21.6724 20.701 677
(L. major): LmjF.13.1090) F10 149 LLHDRQHSI 73 66.66% 72.7166 - 87
F11 187 GLEQQIQEI 81 66.66% - - 623
F12 150 MLQTNSLAL 85 61.11% 36.3161 24.802 1677
G1 24 SLQFSAFLL 68 58.33% 123.9019 21.087 2809
G2 117 FMEVFGMLV 50 52.77% 56.1955 - 148
Pr 78 G3 83 MLVQSCTSI 90 55.55% - - 1781
G4 110 VVSVLTHSV 69 58.33% - - 2390
G5 106 WIPPVVSVL 56 66.66% - - 5645
Pr 90 (Ribosomal G6 286 KIYQIGRSV 61 61.11% 21.4220 - 352
protein L3: LmjF.32.3130) G7 282 QLNKKIYQI 88 69.44% 23.9954 - -
Open in a new tab

a: Results expressed as score.

b: Results expressed as percentage calculated according to the highest score (= 36).

Fig 1. Binding affinity of LmES peptides to HLA-A*0201 molecules.

Fig 1

Open in a new tab

The affinities of selected peptides were determined by class I HLA stabilization assay. (A) Results for individual peptides. T2 cells were initially incubated with 100μg (final concentration) of each of the peptides/mL for 16h at 37°C, followed by incubation at 37°C for 2h in presence of Brefeldine A. HLA-A2 expression on these cells was analyzed by flow cytometry using the BB7.2 antibody. MHC stabilization efficiency for each peptide was calculated as the percentage increase of the mean fluorescence above that of the negative controls. Results were expressed as relative fluorescence intensity (RFI). (B) Box plot results. ++: RFI ranges from 200 to 300%, +: RFI ranges from 100 to 200%, and -: RFI ranges from 0 to 100%.

Stimulation with peptide pools induces production of GrB

Given their large number and to test their immunogenicity in vitro, the predicted peptides were compiled into 20 pools as shown in Table 3. Each pool contains peptides belonging to the same protein. Pools were tested for their ability to induce GrB secretion by PBMCs obtained from 5 HLA-A*0201+-immune donors and 2 HLA-A*0201+ healthy donors. Surprisingly, low IFN-γ levels, not exceeding 45pg/mL (quantification threshold), were detected in culture supernatants of PBMCs obtained from immune individuals, stimulated with the different peptide pools. Similar results were obtained for IL-10, which was detected at low levels (ranging from 20 to 120pg/mL) in only one immune individual. Stimulation of PBMCs from these individuals with SLA or PMA/Ionomycin showed high levels of IFN-γ (data not shown).

Table 3. Setup of peptide pools.

Protein 9.1 9.2 12 13 15 20.1 20.2 22 22.1 27 31 37 38 57 66 68 74 77 78 90
Pool* 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
A1 A7 A8 B1 B7 C1 C6 C9 C11 D4 D5 D12 E3 E8 E10 E11 F4 F7 F12 G6
A2 A9 B2 B8 C2 C7 C10 C12 D6 E1 E4 E9 E12 F5 F8 G1 G7
A3 A10 B4 B9 C3 C8 D1 D7 E2 E5 F1 F6 F9 G2
A4 A11 B5 B10 C4 D2 D8 E6 F2 F10 G3
A5 A12 B6 B11 C5 D3 D9 E7 F3 F11 G4
A6 B12 D10 G5
D11
n 6 1 5 5 6 5 3 2 5 1 7 3 5 2 1 5 3 5 6 2
Open in a new tab

* Numbers 1 to 20 refer to peptide pools.

n: Number of peptides made up for each protein.

As shown in Fig 2A, peptide pools induce variable levels of GrB in culture supernatants of PBMCs obtained from immune individuals. In contrast, for healthy donors no GrB production could be induced. Considering the variability of detected GrB levels, we have resorted to a ranking method (Fig 2C). Classification of the 20 peptide pools corresponding to the 20 different LmES proteins was done according to their capacity to induce GrB production. The concept consists of computing the rank of the different pools for each individual and then calculating the mean rank of each pool for the 5 individuals tested. Interestingly, the Kruskal-Wallis test has revealed that the highest GrB levels were induced by the peptide pools P19, P20, P13, P18, P12, and P17 corresponding respectively to the proteins Pr78, Pr90, Pr38, Pr77, Pr37, and Pr74. GrB levels measured in culture supernatants of PBMCs stimulated with these peptide pools were significantly higher compared to those induced by the other ones (p = 0.0002). Taken together, these results allow us to rank 6 proteins among the potentially ES proteins as best generators of peptides that are recognized by PBMCs of HLA-A*0201+-immune individuals.

Fig 2. Peptides grouped in pools induced GrB production.

Fig 2

Open in a new tab

(A) PBMCs from 5 HLA-A*0201+-donors with a history of ZCL in response to stimulation with peptide pools at a final concentration of 1μg/mL per peptide or (B) SLA (10μg/mL) and PMA/Ionomycin (10ng/mL and 50ng/mL, respectively) as positive controls. GrB production was assessed in culture supernatants using ELISA. (C) Rank of the peptide pools. +: mean pool rank, -: median.

Evaluation of GrB and IFN-γ production by PBMCs stimulated with individual peptides

All peptides belonging to the selected proteins were tested separately for their capacity to induce GrB and IFN-γ. PBMCs obtained from 3 HLA-A*0201+ and 3 HLA-A*0201—immune donors were stimulated with the different individual peptides, then GrB and IFN-γ levels were measured in culture supernatants. We have used PBMCs obtained from 2 HLA-A*0201+-healthy individuals as negative controls. It should first be mentioned that levels of IFN-γ detected in culture supernatants were very weak (< 45pg/mL) in the individuals tested with most of the peptides. Regarding GrB, no production could be detected in supernatants of PBMCs obtained from negative controls in response to stimulation with any of these peptides (data not shown). Interestingly, variable levels of GrB were detected in HLA-A*0201+ and HLA-A*0201—immune donors (Fig 3A). However, there was no statistically significant difference between HLA-A*0201+ and HLA-A*0201—immune individuals (p > 0.05 for all tested peptides). Taken together, 6 peptides (E2, E6, F6, G2, G3, and G4) among the 24 tested have been shown to induce the highest levels of GrB (Fig 3B). Furthermore, 5 peptides out of the 6 selected ones stabilized HLA-A2 molecule on T2 cells with high (F6, G2, and G3) or intermediate (E2 and G4) affinity. Only E6 showed no affinity for HLA-A2 molecule.

Fig 3. GrB induction by the selected peptides.

Fig 3

Open in a new tab

(A) PBMCs from HLA-A*0201+ (black mark) and HLA-A*0201- (white mark) -healed ZCL individuals were stimulated with selected peptides separately. The supernatant was collected after 5 days of incubation and assayed for GrB production. (B) Peptides E2, E6, F6, G2, G3, and G4 induced the highest levels of GrB.

Discussion

For a long time, it has been a consensus that a Th1 dominant response promotes IFN-γ production, induces lesion healing, and controls parasite burden [7]. Based on this, different vaccine candidates have been selected. CD8+ T cells play a major role in controlling leishmaniasis, since growing evidence did prove their participation in the immune response against different Leishmania species studied in experimental models and humans [29, 30]. Few studies have focused on the identification of Leishmania epitopes that can be presented by class I MHC molecules to CD8+ T cells [31, 32]. Currently, there are no well-defined Leishmania CD8+ T cell epitopes, which has made it difficult to investigate how CD8+ T cell activation occurs in leishmaniasis. Antigen-presenting cells, such as macrophages and dendritic cells have been shown to be able to capture, process, and present in a class I MHC-restricted manner various exogenous antigens including those derived from intracellular pathogens like Leishmania parasites [17, 33].

Previously, we have characterized 33 Leishmania genes coding for proteins that are probably released by the parasite in the phagolysosomal vacuole [20].

Herein, we have analyzed these potentially LmES proteins in an attempt to identify HLA-A*0201-binding peptides able to activate CD8+ T cells. We have identified 6 epitopes: E2, E6, F6, G2, G3, and G4 that are able to induce GrB production by PBMCs obtained from immune individuals. These peptides derived from the sequence of the Pr37, Pr38, Pr78, and Pr74 proteins. Our study is not exhaustive since the choice of the 33 potentially ES protein sequences was made out of more than 8,000 parasite protein-coding genes. In fact, there are probably additional Leishmania ES proteins that have not been described as of yet. Moreover, it is quite possible that non-excreted parasitic antigens able to generate CD8+ T cell epitopes do also exist.

Our hypothesis is clear and our approach is simple. We have assumed that LmES proteins may generate peptides that could be presented to CD8+ T cells. This approach oriented us towards 4 proteins of interest. Pr74 corresponding to elongation factor-1 alpha (EF-1α), which is a multifunctional protein essentially involved in protein biosynthesis and parasite survival in infected macrophages [34–39]. Indeed, the presence of Leishmania EF-1α in the cytosol of infected macrophages has also been demonstrated [37, 38]. Interestingly, this protein was one of the leishmanial antigens that was used for construction of the vaccine LeishDNAvax composed of MIDGE-TH1 vectors encoding 5 conserved leishmanial antigens: KMP11, TSA, CPA, CPB, and Pr74 [40]. The 3 remaining proteins were described as potentially secreted by the parasite but do not correspond to any proteins described in sequence libraries. Among them, 2 proteins Pr37 and Pr78 contain signal peptide sequences and one, Pr38 was predicted to be secreted via non-classical pathways [20].

In this study, the selection of peptides was performed using the computer-based prediction method, which constitutes a useful tool for peptide identification. However, this method is unable to predict all peptide sequences. Thus, some interesting and immunogenic peptides could be missed out and prevented from being tested in the immune response, only because in silico methods could not predict them. So the best way is to use overlapping peptides to scan all protein sequences as performed by Basu and collaborators regarding peptide identification belonging to the protein Kmp11 [31]. Nonetheless, this method could not be applied in our study because it would have given us thousands of peptides to test, which was currently not feasible for all proteins tested here. Moreover, Pelte and collaborators have previously identified one single stimulating peptide, which did not stabilize HLA-A*0201 expression on T2 cells and could therefore not be presented by HLA-A*0201 [41]. The paradox of T cell recognition of a peptide that fails to bind to HLA-A2 could be explained by the fact that peptides could be recognized after binding to other class I HLA molecules carried out by the patients, which could subsequently present the epitope to specific T cells.

The next step was to analyse the immunogenicity of the antigenic peptides in naturally-infected individuals. In addition to their capacity to bind to class I MHC molecules, we have assumed that these peptides exist in large quantities in the intracellular phagolysosomal vesicle. Consequently, in natural infection some peptides predicted to have high affinity in theoretical and functional tests could fail to induce significant immune response since they are not secreted or because they are in different cellular structures. By contrast, some low-affinity peptides can still be presented by class I MHC molecules because of their abundance in the intracellular phagolysosomal vesicle. For these reasons, all predicted peptides were compiled in pools and their immunogenicity tested in HLA-A*0201+-ZCL recovered individuals. Pooling peptides has been used in many previous studies [28, 32] and does not seem to be a limiting factor [42, 43] considering that all peptides are predicted with almost equal affinity for HLA binding and with same stimulatory concentrations in cultures. Unexpectedly, weak levels of IFN-γ were detected in culture supernatants of PBMCs stimulated with the different peptide pools. This cannot be attributed to the inhibition of IFN-γ production by IL-10, which was not detected in these culture supernatants. This could rather be explained by the possibility of low frequency of memory CD8+ T cells due to stimulation conditions. In fact, in the present study PBMCs were stimulated with peptide pools without adding IL-2 or anti-CD48 as done by Seyed and collaborators [32]. Our results are similar to those described in other studies using different read out systems for the IFN-γ detection in T cells, such as flow cytometry [41] or ELISPOT [28]. Results of these two studies showed a weak production of IFN-γ induced by only few peptides among those selected by using bioinformatics.

Further, we have shown here that variable levels of GrB were induced by the different peptide pools, which led us to rely on the ranking method. Thus, we have selected 6 proteins as the best generators of peptides recognized by PBMCs obtained from HLA-A*0201-immune individuals. Consequently, we have analyzed separately the immunogenicity of all peptides belonging to these proteins. The highest GrB levels were detected in supernatants of PBMCs stimulated with the peptides E2, E6, F6, G2, G3, and G4. Unexpectedly, these peptides have also induced GrB production in HLA-A*0201-negative immune individuals. Similar results have been reported by Seyed and collaborators [32]. As discussed by the authors, this could be explained by specificity overlap between supertypes of HLA molecules and would need to be further confirmed in a larger population of individuals bearing other HLA-A alleles [32, 44]. To achieve that, we will be extending our study to map potential CD8+ T cell epitopes restricted to other common class I HLA alleles.

To better trigger the specific response to our peptides, several experiments are planned, i.e., establishing “short-term” cell lines specific of the selected peptides and analyzing their ability to induce the production of GrB, IFN-γ, IL-2, and IL-10 when co-cultured in the presence of the T2 cell line pulsed with each of the peptides, and used as antigen-presenting cells.

In conclusion, we have identified novel HLA-A*0201-restricted immunogenic CD8+ T cell epitopes derived from potentially LmES proteins using in silico prediction and functional studies on PBMCs obtained from immune individuals. Proteins we have identified here could constitute potential candidate vaccine antigens.

Acknowledgments

We are grateful to the volunteers who participated in the study and the team working at the endemic sites that allowed the achievement of this work. We are also thankful to Dr. Salem Chouaib (Gustave Roussy Institute, Villejuif, France) for providing us with the T2 cell line. We also thank Ms. Beya Larguech (Pasteur Institute of Tunis) for her technical assistance with the flow cytometric analyses.

Data Availability

All relevant data are within the paper.

Funding Statement

This work was supported by P50AI074178 from the National Institute of Allergy and Infection diseases to HL and ABS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

  • 1.Hotez P, Ottesen E, Fenwick A, Molyneux D. The neglected tropical diseases: the ancient afflictions of stigma and poverty and the prospects for their control and elimination. Adv Exp Med Biol. 2006;582:23–33. . [DOI] [PubMed] [Google Scholar]
  • 2.Choi CM, Lerner EA. Leishmaniasis: recognition and management with focus on the immunocompromised patient. Am J Clin Dermatol. 2002;3(2):91–105. . [DOI] [PubMed] [Google Scholar]
  • 3.Reiner SL, Locksley RM. The regulation of immunity to Leishmania major. Annu Rev Immunol. 1995;13:151–77. . [DOI] [PubMed] [Google Scholar]
  • 4.Mansueto P, Vitale G, Di Lorenzo G, Rini GB, Mansueto S, Cillari E. Immunopathology of leishmaniasis: an update. Int J Immunopathol Pharmacol. 2007;20(3):435–45. . [DOI] [PubMed] [Google Scholar]
  • 5.Piscopo TV, Mallia AC. Leishmaniasis. Postgrad Med J. 2006;82(972):649–57. . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Murray HW, Berman JD, Davies CR, Saravia NG. Avances in leishmaniasis. Lancet. 2005;366(9496):1561–77. . [DOI] [PubMed] [Google Scholar]
  • 7.Nylén S, Gautam S. Immunological perspectives of leishmaniasis. J Glob Infect Dis. 2010;2(2):135–46. 10.4103/0974-777X.62876 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Tripathi P, Singh V, Naik S. Immune response to Leishmania: paradox rather than paradigm. FEMS Immunol Med Microbiol. 2007;51(2):229–42. . [DOI] [PubMed] [Google Scholar]
  • 9.Roberts MT. Current understandings on the immunology of leishmaniasis and recent developments in prevention and treatment. Br Med Bull. 2006;75–76:115–30. . [DOI] [PubMed] [Google Scholar]
  • 10.Carvalho EM, Johnson WD, Barreto E, Marsden PD, Costa JL, Reed S, et al. Cell mediated immunity in American cutaneous and mucosal leishmaniasis. J Immunol. 1985;135(6):4144–8. . [PubMed] [Google Scholar]
  • 11.Castes M, Agnelli A, Verde O, Rondón AJ. Characterization of the cellular immune response in American cutaneous leishmaniasis. Clin Immunol Immunopathol. 1983;27(2): 176–86. . [DOI] [PubMed] [Google Scholar]
  • 12.Kemp M, Hey AS, Kurtzhals JA, Christensen CB, Gaafar A, Mustafa MD, et al. Dichotomy of the human T cell response to Leishmania antigens. I. Th1-like response to Leishmania major promastigote antigens in individuals recovered from cutaneous leishmaniasis. Clin Exp Immunol. 1994;96(3):410–15. . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Bousoffara T, Louzir H, Ben Salah A, Dellagi K. Analysis of granzyme B activity as a surrogate marker of Leishmania-specific cell-mediated cytotoxicity in zoonotic cutaneous leishmaniasis. J Infect Dis. 2004;189(7):1265–73. . [DOI] [PubMed] [Google Scholar]
  • 14.Faria DR, Souza PE, Durães FV, Carvalho EM, Gollob KJ, Machado PR, et al. Recruitment of CD8+ T cells expressing granzyme A is associated with lesion progression in human cutaneous leishmaniasis. Parasite Immunol. 2009;31(8):432–9. 10.1111/j.1365-3024.2009.01125.x [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Barral-Netto M, Barral A, Brodskyn C, Carvalho EM, Reed SG. Cytotoxicity in human mucosal and cutaneous leishmaniasis. Parasite Immunol. 1995;17(1):21–8. . [DOI] [PubMed] [Google Scholar]
  • 16.Brodskyn CI, Barral A, Boaventura V, Carvalho E, Barral-Netto M. Parasite-driven in vitro human lymphocyte cytotoxicity against autologous infected macrophages from mucosal leishmaniasis. J Immunol. 1997;159(9):4467–73. . [PubMed] [Google Scholar]
  • 17.Bertholet S, Debrabant A, Afrin F, Caler E, Mendez S, Tabbara KS, et al. Antigen requirements for efficient priming of CD8+ T cells by Leishmania major-infected dendritic cells. Infect Immun. 2005;73(10):6620–8. . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Bertholet S, Goldszmid R, Morrot A, Debrabant A, Afrin F, Collazo-Custodio C, et al. Leishmania antigens are presented to CD8+ T cells by a transporter associated with antigen processing-independent pathway in vitro and in vivo. J Immunol. 2006;177(6):3525–33. . [DOI] [PubMed] [Google Scholar]
  • 19.Kima PE, Ruddle NH, McMahon-Pratt D. Presentation via the class I pathway by Leishmania amazonensis-infected macrophages of an endogenous leishmanial antigen to CD8+ T cells. J Immunol. 1997;159(4):1828–34. . [PubMed] [Google Scholar]
  • 20.Chenik M, Lakhal S, Ben Khalef N, Zribi L, Louzir H, Dellagi K. Approaches for the identification of potential excreted/secreted proteins of Leishmania major parasites. Parasitology. 2006;132(Pt 4):493–509. . [DOI] [PubMed] [Google Scholar]
  • 21.Ayed K, Jendoubi SA, Makhlouf M, Sfar I, Abdallah TB. Comparison of HLA Class I and II molecular and serological typing within clinical laboratory. Saudi J Kidney Dis Transpl. 2003;14(1):39–42. . [PubMed] [Google Scholar]
  • 22.Daryani A, Hosseini AZ, Dalimi A. Immune responses against excreted/secreted antigens of Toxoplasma gondii tachyzoites in the murine model. Vet Parasitol. 2003;113(2):123–34. . [DOI] [PubMed] [Google Scholar]
  • 23.Mustafa AS. Development of new vaccines and diagnostic reagents against tuberculosis. Mol Immunol. 2002;39(1–2):113–19. . [DOI] [PubMed] [Google Scholar]
  • 24.Pym AS, Brodin P, Majlessi L, Brosch R, Demangel C, Williams A, et al. Recombinant BCG exporting ESAT-6 confers enhanced protection against tuberculosis. Nat Med. 2003;9(5):533–9. . [DOI] [PubMed] [Google Scholar]
  • 25.Shams H, Klucar P, Weis SE, Lalvani A, Moonan PK, Safi H, et al. Characterization of a Mycobacterium tuberculosis peptide that is recognized by human CD4+ and CD8+ T cells in the context of multiple HLA alleles. J Immunol. 2004;173(3):1966–77. . [DOI] [PubMed] [Google Scholar]
  • 26.Naouar I, Boussoffara T, Ben Ahmed M, Belhaj Hmida N, Gharbi A, Gritli S, et al. Involvement of different CD4+ T cell subsets producing granzyme B in the immune response to Leishmania major antigens. Mediators Inflamm. 2014:63609 10.1155/2014/636039 . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Gomez-Nunez M, Pinilla-Ibarz J, Dao T, May RJ, Pao M, Jaggi JS, et al. Peptide binding motif predictive algorithms correspond with experimental binding of leukemia vaccine candidate peptides to HLA-A*0201 molecules. Leuk Res. 2006;30(10):1293–8. . [DOI] [PubMed] [Google Scholar]
  • 28.Elkington R, Walker S, Crough T, Menzies M, Tellam J, Bharadwaj M, et al. Ex vivo profiling of CD8+ T-cell responses to human cytomegalovirus reveals broad and multispecific reactivities in healthy virus carriers. J Virol. 2003;77(9):5226–40. . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Stäger S, Rafati S. CD8+ T cells in Leishmania infections: friends or foes? Front Immunol. 2012;3:5 10.3389/fimmu.2012.00005 eCollection 2012. . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Novais FO, Scott P. CD8+ T cells in cutaneous leishmaniasis: the good, the bad, and the ugly. Semin Immunopathol. 2015;37(3):251–9. 10.1007/s00281-015-0475-7 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Basu R, Roy S, Walden P. HLA class I-restricted T cell epitopes of the kinetoplastid membrane protein-11 presented by Leishmania donovani infected human macrophages. J Infect Dis. 2007;195(9):1373–80. . [DOI] [PubMed] [Google Scholar]
  • 32.Seyed N, Zahedifard F, Safaiyan S, Gholami E, Doustdari F, Azadmanesh K, et al. In silico analysis of six known Leishmania major antigens and in vitro evaluation of specific epitopes eliciting HLA-A2-restricted CD8+ cell response. PLOS Negl Trop Dis. 2011;5:e1295 10.1371/journal.pntd.0001295 . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Houde M, Bertholet S, Gagnon E, Brunet S, Goyette G, Laplante A, et al. Phagosomes are competent organelles for antigen cross-presentation. Nature. 2003;425(6956):402–6. . [DOI] [PubMed] [Google Scholar]
  • 34.Browne GJ, Proud CG. Regulation of peptide-chain elongation in mammalian cells. Eur J Biochem. 2002;269(22):5360–8. . [DOI] [PubMed] [Google Scholar]
  • 35.Condeelis J. Elongation factor 1 alpha, translation and the cytoskeleton. Trends Biochem Sci. 1995;20(5):169–70. . [DOI] [PubMed] [Google Scholar]
  • 36.Nandan D, Cherkasov A, Sabouti R, Yi T, Reiner NE. Molecular cloning, biochemical and structural analysis of elongation factor-1 alpha from Leishmania donovani: comparison with the mammalian homologue. Biochem Biophys Res Commun. 2003;302(4):646–52. . [DOI] [PubMed] [Google Scholar]
  • 37.Nandan D, Reiner NE. Leishmania donovani engages in regulatory interference by targeting macrophage protein tyrosine phosphatase SHP-1. Clin Immunol. 2005;114(3): 266–77. . [DOI] [PubMed] [Google Scholar]
  • 38.Nandan D, Yi T, Lopez M, Lai C, Reiner NE. Leishmania EF-1 alpha activates the Src homology 2 domain containing tyrosine phosphatase SHP-1 leading to macrophage deactivation. J Biol Chem. 2002;277(51):50190–7. . [DOI] [PubMed] [Google Scholar]
  • 39.Ryazanov AG, Rudkin BB, Spirin AS. Regulation of protein synthesis at the elongation stage. New insights into the control of gene expression in eukaryotes. FEBS Lett. 1991;285(2):170–75. . [DOI] [PubMed] [Google Scholar]
  • 40.Das S, Freier A, Boussoffara T, Das S, Oswald D, Losch FO, et al. Modular multiantigen T cell epitope-enriched DNA vaccine against human leishmaniasis. Sci Transl Med. 2014;6(234):234ra56 10.1126/scitranslmed.3008222 . [DOI] [PubMed] [Google Scholar]
  • 41.Pelte C, Cherepnev G, Wang Y, Schoenemann C, Volk HD, Kern F. Random screening of proteins for HLA-A*0201-binding nine-amino acid peptides is not sufficient for identifying CD8+ T cell epitopes recognized in the context of HLA-A*0201. J Immunol. 2004;172(11):6783–9. . [DOI] [PubMed] [Google Scholar]
  • 42.Suneetha PV, Schlaphoff V, Wang C, Stegmann KA, Fytili P, Sarin SK, et al. Effect of peptide pools on effector functions of antigen-specific CD8+ T cells. J Immunol Methods. 2009;342(1–2):33–48. 10.1016/j.jim.2008.11.020 [DOI] [PubMed] [Google Scholar]
  • 43.Lyke KE, Burges RB, Cissoko Y, Sangare L, Kone A, Dao M, et al. HLA-A2 supertype-restricted cell-mediated immunity by peripheral blood mononuclear cells derived from Malian children with severe or uncomplicated Plasmodium falciparum malaria and healthy controls. Infect Immun. 2005;73(9):5799–808. . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Sidney J, Peters B, Frahm N, Brander C, Sette A. HLA class I supertypes: a revised and updated classification. BMC Immunol. 2008;9:1 10.1186/1471-2172-9-1 . [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

All relevant data are within the paper.

Articles from PLoS ONE are provided here courtesy of PLOS

RESOURCES