# Design of multi-epitope peptides containing HLA class-I and class-II-restricted epitopes derived from immunogenic Leishmania proteins, and evaluation of CD4+ and CD8+ T cell responses induced in cured cutaneous leishmaniasis subjects

Sarra Hamrouni, Rachel Bras-Gonçalves, Abdelhamid Kidar, Karim Aoun, Rym Chamakh-Ayari, Elodie Petitdidier, Yasmine Messaoudi, Julie Pagniez, Jean-Loup Lemesre, Amel Meddeb-Garnaoui

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Sarra Hamrouni, Rachel Bras-Gonçalves, Abdelhamid Kidar, Karim Aoun, Rym Chamakh-Ayari, et al. Design of multi-epitope peptides containing HLA class-I and class-II-restricted epitopes derived from immunogenic Leishmania proteins, and evaluation of CD4+ and CD8+ T cell responses induced in cured cutaneous leishmaniasis subjects. PLoS Neglected Tropical Diseases, 2020, 14 (3), pp.e0008093. 10.1371/journal.pntd.0008093. pasteur-03557433

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RESEARCH ARTICLE

# Design of multi-epitope peptides containing HLA class-I and class-II-restricted epitopes derived from immunogenic *Leishmania* proteins, and evaluation of CD4+ and CD8+ T cell responses induced in cured cutaneous leishmaniasis subjects

Sarra Hamrouni<sup>1,2,3</sup>, Rachel Bras-Gonçalves<sup>1,3</sup>, Abdelhamid Kidar<sup>4</sup>, Karim Aoun<sup>1</sup>, Rym Chamakh-Ayari<sup>1,2</sup>, Elodie Petitdidier<sup>1,3</sup>, Yasmine Messaoudi<sup>1,2,3</sup>, Julie Pagniez<sup>3</sup>, Jean-Loup Lemesre<sup>1,3</sup>, Amel Meddeb-Garnaoui<sup>1*</sup>

1 Laboratoire de Parasitologie Médicale, Biotechnologie et Biomolécules, Institut Pasteur de Tunis, Tunis, Tunisie, 2 Faculté des Sciences de Bizerte, Université de Carthage, Tunis, Tunisie, 3 UMR INTERTRYP, Université de Montpellier, IRD, CIRAD, Montpellier, France, 4 Hôpital Régional de Gafsa, Gafsa, Tunisia

* amel.garnaoui@pasteur.tn

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OPEN ACCESS

**Citation:** Hamrouni S, Bras-Gonçalves R, Kidar A, Aoun K, Chamakh-Ayari R, Petitdidier E, et al. (2020) Design of multi-epitope peptides containing HLA class-I and class-II-restricted epitopes derived from immunogenic *Leishmania* proteins, and evaluation of CD4+ and CD8+ T cell responses induced in cured cutaneous leishmaniasis subjects. PLoS Negl Trop Dis 14(3): e0008093. https://doi.org/10.1371/journal.pntd.0008093

**Editor:** Mary Ann McDowell, University of Notre Dame, UNITED STATES

**Received:** July 9, 2019

**Accepted:** January 27, 2020

**Published:** March 16, 2020

**Copyright:** © 2020 Hamrouni 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.

**Data Availability Statement:** All relevant data are within the manuscript and its Supporting Information files.

**Funding:** This work was supported by Institut de Recherche pour le développement (IRD). A. M-G is the recipient of 'Jeune Equipe associée à l'IRD' (JEAI-VACLish). The funder had no role in study

## Abstract

Human leishmaniasis is a public health problem worldwide for which the development of a vaccine remains a challenge. T cell-mediated immune responses are crucial for protection. Peptide vaccines based on the identification of immunodominant T cell epitopes able to induce T cell specific immune responses constitute a promising strategy. Here, we report the identification of human leukocyte antigen class-I (HLA-I) and -II (HLA-II)-restricted multi-epitope peptides from *Leishmania* proteins that we have previously described as vaccine candidates. Promastigote Surface Antigen (PSA), LmIRAB (L. major large RAB GTPase) and Histone (H2B) were screened, in silico, for T cell epitopes. 6 HLA-I and 5 HLA-II-restricted multi-epitope peptides, able to bind to the most frequent HLA molecules, were designed and used as pools to stimulate PBMCs from individuals with healed cutaneous leishmaniasis. IFN-γ, IL-10, TNF-α and granzyme B (GrB) production was evaluated by ELISA/CBA. The frequency of IFN-γ-producing T cells was quantified by ELISpot. T cells secreting cytokines and memory T cells were analyzed by flow cytometry. 16 of 25 peptide pools containing HLA-I, HLA-II or HLA-I and -II peptides were able to induce specific and significant IFN-γ levels. No IL-10 was detected. 6 peptide pools were selected among those inducing the highest IFN-γ levels for further characterization. 3/6 pools were able to induce a significant increase of the percentages of CD4+IFN-γ+, CD8+IFN-γ+ and CD4+GrB+ T cells. The same pools also induced a significant increase of the percentages of bifunctional IFN-γ+/TNF-α+CD4+ and/or central memory T cells. We identified highly promiscuous HLA-I and -II restricted epitope combinations from H2B, PSA and LmIRAB proteins that stimulate both CD4+ and CD8+ T cell responses in recovered individuals. These multi-epitope peptides could be used as potential components of a polytope vaccine for human leishmaniasis.
design, data collection and analysis, decision to
publish, or preparation of the manuscript.

**Competing interests:** The authors have declared
that no competing interests exist.

Author summary

The control of leishmaniasis, a neglected tropical disease of public health importance, caused by protozoan parasites of the genus *Leishmania*, mainly relies on chemotherapy, which is highly toxic. Currently, there is no vaccine against human leishmaniasis. Peptide-based vaccines consisting of T cell epitopes identified within proteins of interest by epi-tope predictive algorithms are a promising strategy for vaccine development. Here, we identified multi-epitope peptides composed of HLA-I and -II-restricted epitopes, using immunoinformatic tools, within *Leishmania* proteins previously described as potential vaccine candidates. We showed that multi-epitope peptides used as pools were able to activate IFN-γ producing CD4+ as well as CD8+ T cells, both required for parasite elimi-nation. In addition, granzyme B-producing CD4+ T cells, bifunctional CD4+ IFN-γ +/TNF-α+ and/or TNF-α+/IL-2+ T cells as well as CD4+ and CD8+ central memory T cells, all involved in *Leishmania* infection control, were significantly increased in response to multi-epitope peptide stimulation. As far as we know, no study has described the detec-tion of both CD4+ and CD8+ T cell populations in response to stimulation by both HLA-I and II-restricted peptides in humans. The immunogenic HLA-I and -II-restricted multi-epitope peptides identified in this study could constitute potential vaccine candidates against human leishmaniasis.

**Introduction**

Leishmaniasis is caused by an intracellular parasite of the *Leishmania* genus. It is a severely
neglected tropical disease associated with considerable morbidity and mortality throughout
the world. This disease transmitted by sand fly bites can have a wide spectrum of clinical mani-
festations ranging from self-healing cutaneous lesions to fatal visceral disease, depending on
the infecting parasite species, the host immune response and the sand fly saliva components
[1, 2]. Cutaneous leishmaniasis (CL) is the most frequent form with 0.7–1 million new cases
occurring annually worldwide [1, 3]. Zoonotic CL, caused by *Leishmania* (L.) major, is
endemic in Tunisia, with thousands of cases reported every year [4, 5]. The control of leish-
maniasis mainly relies on chemotherapy, which is highly toxic and contributes to the emer-
gence of drug-resistant parasites [6]. Most individuals who recover from leishmaniasis develop
lifelong immunity to re-infection suggesting that a vaccine is feasible. It is generally considered
that a dominant T helper 1 (Th1)-type cellular immune response and IFN-γ production are
critical for infection control. In the murine model of *L. major* infection, CD4+ Th1 cells secrete
IFN-γ and TNF-α, leading to the parasite elimination by activated macrophages, whereas,
CD4+ Th2 response producing IL-4 and IL-13 favors disease progression [7, 8]. Th1/Th2
dichotomy is absent in human leishmaniasis. It is now clear that the immune response against
*Leishmania* parasites is more complex both in humans and mice [9]. In human infection, Th1
CD4+ T cells producing IFN-γ and TNF-α and positive delayed type hypersensitivity (DTH)
responses, have been associated with the healing process [10–14]. IL-10 was associated with a
lack of parasite control but may also play a role in the control of excessive inflammatory
response [15–19]. The induction of multifunctional Th1 cells secreting IFN-γ, TNF-α and IL-
2, has been described to correlate with protection [20–23]. CD8+ T cells are also important in
the healing mainly through IFN-γ production [24–26]. These cells have also been involved in
pathogenesis, trough Granzyme B (GrB) production [27–30], while other studies showed that
an increase of GrB activity was associated with a good prognosis in patients with CL [31–33].
Both central (TCM) and effector memory T cells (TEM) have been characterized in human CL and could play a role in protection against *Leishmania* infection [34–36]

Several vaccination strategies against leishmaniasis have been examined so far including leishmanization, killed or attenuated parasites, DNA and subunits vaccines including native or recombinant proteins [37, 38]. However, there is currently no vaccine for humans. In recent years, the use of peptides containing the minimal immunogenic part of a protein capable of inducing a desired specific T cell response may become a promising strategy in leishmaniasis prophylaxis [39–41]. In addition, the development of bioinformatic tools has made it easier to identify potential immunogenic Human Leukocyte Antigens (HLA)-restricted T cell epitopes for vaccines. Peptide-based vaccines have many advantages including absence of infectious materials, stability, specificity and large-scale production at low cost. Peptide-based vaccines have been successfully tested against cancer and infectious diseases [42–46]. Potential immunogenic peptides can be identified within proteins that have been previously described as vaccine candidates. We already reported that *Leishmania* histone H2B, Promastigote Surface Antigen (PSA) and *L. major* large RAB GTPase (LmlRAB), were able to induce a predominant Th1 response in individuals immune to *L. major* or *L. infantum* [47–49]. These proteins are involved in the organization and function of DNA within the nucleus for histone, and in resistance to complement lysis and macrophage invasion for PSA, whereas a role in phagosome maturation and regulation of the secretory pathway was described for RAB proteins in *Leishmania* parasites. H2B, PSA and LmlRAB are highly conserved among *Leishmania* species while displaying low level of homology with mammalian homologues. They are present in both amastigote and promastigote stages and confer significant protection in mice and dogs [47–52].

Here, we identify potential immunogenic multi-epitope peptides from H2B, PSA and LmlRAB *Leishmania* proteins. Promiscuous T cell epitopes able to bind to several HLA class I (HLA-I) or HLA class II (HLA-II) molecules were identified using online epitope prediction software tools and designed to construct multi-epitope peptides. Their capacity to stimulate cytokine-producing CD4, CD8, multifunctional and memory T cells was evaluated, in vitro, in individuals with healed CL due to *L. major*.

## Materials and methods

### Ethics statement

The recruitment and sampling collection of groups of volunteers were conducted with the approval of the local ethical committee of the Pasteur Institute of Tunis (protocol number 2015/01/I/LR11IPT06). All subjects analyzed were over 18 years old. A written informed consent was obtained from all subjects before enrollment.

### Study population

Human groups included cured CL and healthy individuals (Table 1). Cured individuals were recruited from well-characterized endemic areas for CL due to *L. major* located in central

<table><caption>Table 1. Study population.</caption><thead><tr><td>Status</td><td>Cured from CL</td><td>Healthy</td></tr></thead><tbody><tr><td>Number of individuals</td><td>61</td><td>28</td></tr><tr><td>Average age (year) (Mean±SD)</td><td>44.6±14.5</td><td>32±11.7</td></tr><tr><td>Sex ratio (M/F)</td><td>0.48</td><td>0.3</td></tr><tr><td>Average LCZ scar number (Mean±SD)</td><td>1.8±1.1</td><td>0</td></tr><tr><td>IFN-γ response to SLA (pg/ml)*</td><td>2298±1689.9/219.9±843</td><td>18.9±19.5/64.9±137.7</td></tr></tbody></table>

<footnote>*Mean of SLA ± SD/Mean of unstimulated ± SD</footnote>

<footnote>https://doi.org/10.1371/journal.pntd.0008093.t001</footnote>
Tunisia (Kairouan and Gafsa), based on the following inclusion criteria (i) living in endemic foci to *L. major* (ii) well documented medical records (iii) presence of typical scars and (iv) positive IFN-γ response to Soluble *Leishmania* Antigens (SLA). All patients have recovered after a course of standard Glucantime therapy, with the exception of 3 individuals who have practiced self-medication. Healthy individuals were recruited in a low endemic area (Tunis), with no history of leishmaniasis and no IFN-γ response to SLA. Exclusion criteria were immunosuppressive diseases other than leishmaniasis, long-term treatment and pregnancy. Hepatinized blood was collected from cured CL donors and healthy individuals used as a control group (Table 1).

## HLA typing

DNA was extracted from peripheral blood using QIAamp DNA Blood Mini Kit (QIAGEN GmbH, Germany) according to the manufacturer's instructions. HLA genotyping of DNA samples was performed by Next Generation Sequencing (NGS) (DKMS Life Science Lab, Dresden, Germany).

## Protein selection

For this study, the full sequences of H2B, LmlRAB and PSA proteins derived from multiple *Leishmania* species (*L. major*, *L. infantum*, *L. donovani*, *L. braziliensis*, *L. mexicana*, *L. amazonensis*, *L. chagasi*) were obtained from the national center for biotechnology (NCBI) protein database (http://www.ncbi.nlm.nih.gov/protein/) (Table 2).

Align Sequences Protein BLAST (https://blast.ncbi.nlm.nih.gov/BlastAlign.cgi) and Clustal Omega, a multiple sequence alignment program (https://www.ebi.ac.uk/Tools/msa/clustalo/),

<table><caption>Table 2. *Leishmania* proteins used as candidate antigens for epitope screening.</caption><thead><tr><th>Abbreviations</th><th>Names</th><th>Leishmania species</th><th>Accession number*</th><th>AA** number</th></tr></thead><tbody><tr><td rowspan="5">H2B</td><td rowspan="5">Histone</td><td>L. major</td><td>AF336276</td><td>111</td></tr><tr><td>L. donovani</td><td>XP_003858819</td><td>111</td></tr><tr><td>L. infantum</td><td>XP_001463598</td><td>111</td></tr><tr><td>L. braziliensis</td><td>XP_001564132</td><td>111</td></tr><tr><td>L. mexicana</td><td>XP_003874182</td><td>107</td></tr><tr><td rowspan="10">PSA</td><td rowspan="10">Promastigote Surface Antigen</td><td>L. amazonensis</td><td>ACY70940</td><td>371</td></tr><tr><td>L. infantum</td><td>ACY70941</td><td>463</td></tr><tr><td>L. donovani</td><td>AAY96325</td><td>386</td></tr><tr><td>L. major</td><td>AAB38549</td><td>385</td></tr><tr><td>L. tropica</td><td>AAF80491</td><td>626</td></tr><tr><td>L. braziliensis</td><td>XP_001563125</td><td>912</td></tr><tr><td>L. mexicana</td><td>XP_003873184</td><td>367</td></tr><tr><td>L. chagasi</td><td>AAB62271</td><td>417</td></tr><tr><td rowspan="5">LmlRAB</td><td rowspan="5">Leishmania major large RAB GTPase</td><td>L. major</td><td>AY962589</td><td>611</td></tr><tr><td>L. donovani</td><td>XP_003863179</td><td>609</td></tr><tr><td>L. infantum</td><td>XP_001467349</td><td>609</td></tr><tr><td>L. braziliensis</td><td>XP_001567099.1</td><td>609</td></tr><tr><td>L. mexicana</td><td>XP_003877608.1</td><td>610</td></tr></tbody></table>

<footnote>*The full sequences of the proteins were extracted from NCBI database</footnote>

<footnote>**AA: amino acid</footnote>
were used to analyze sequence similarity between proteins from different *Leishmania* species. For some regions of the proteins that had good homology (but not 100% identical), consensus sequences were obtained with CONS EMBOSS Explorer (http://www.bioinformatics.nl/cgi-bin/emboss/cons) and created according to the proximity of amino acids given by Taylor's classification [53]. Protein BLAST server (https://blast.ncbi.nlm.nih.gov/Blast.cgi) was used to analyze sequence similarity between *Leishmania* and human proteins in order to exclude protein regions with significant homology to human proteins.

## HLA-restricted T cell epitope prediction and design of multi-epitope peptides

To identify HLA-binding epitopes derived from each conserved or consensus protein sequence, NetMHC 3.2 server (http://www.cbs.dtu.dk/services/NetMHC-3.2/), NetMHCII 2.2 Server (http://www.cbs.dtu.dk/services/NetMHCII-2.2/) and IEDB T Cell Epitope Prediction Tools (http://tools.iedb.org/mhci/ and http://tools.iedb.org/mhcii/) were used. For HLA-I-binding epitopes, we have focused on 8, 9 or 10-mer long peptides. Each epitope was predicted for its binding affinity for 57 different HLA molecules (29 HLA-A and 28 HLA-B) considered to be the most frequent in the general worldwide population [54]. These molecules are representative of HLA allele sets, named supertypes, with common repertoires of peptide-binding motifs [55]. The IC50 values to determine the binding affinity of epitopes were calculated by utilizing IEDB and NetMHC 3.2 servers. A threshold of less than 50 nM was used to define strong binders (SB) and less than 500 nM to define weak binders (WB). HLA-I-restricted epitopes derived from each protein were arranged in a string of beads fashion, to construct multi-epitope peptides [56]. To optimize proteasome-mediated processing of these multi-epitope peptides, the proteasomal cleavage sites were predicted by the NetCHOP 3.1 (http://www.cbs.dtu.dk/services/NetChop/) and PAProC II (http://www.paproc2.de/paproc1/paproc2.html) algorithms. Linkers, also known as spacers, composed of some amino acids (ARY, RY, GR or TV), were added to improve peptide proteasomal cleavage for HLA-I presentation, if necessary [57, 58].

For HLA-II-binding epitopes, 15-mer long epitopes were predicted from each conserved or consensus protein sequence. Each epitope was predicted for its binding affinity for the most common HLA-II alleles classified in supertypes main DR (DRB1*0101, DRB1*0701, DRB1*0901, DRB1*1101, DRB1*1201, DRB1*1501, DRB5*0101 and DPB1*1401), DR4 (DRB1*0401, DRB1*0405 and DRB1*0802, DRB3 (DRB1*0301, DRB1*0301, DRB1*0301, 1302, DRB3*0101, DRB3*0202 and DRB4*0101), main DP (DPB1*0101, DPB1*0402 and DPB1*0501), and DP2 (DPB1*0201, and DPB1*0401) [59]. The IC50 values were calculated by utilizing IEDB and NetMHCII 2.2 servers. Hot spot areas of predicted HLA-II-restricted epitopes were selected within each conserved or consensus protein sequence to design long peptides. Global population coverage was estimated from a vaccine candidate composed with all designed multi-epitope peptides, by the IEDB Population coverage tools (http://tools.iedb.org/ population/). Population coverage calculation was performed from 115 countries and 21 different ethnicities grouped into 16 different geographical areas.

## Physico-chemical properties and synthesis of Multi-epitope peptides

ExPASy ProtParam tool (https://web.expasy.org/protparam/) and Peptide property calculator (https://www.pepcalc.com/) were used to evaluate the physicochemical properties of the designed peptides such as hydrophobicity. The molecular weight of multi-epitope peptides was obtained by mass-spectrometry (Proteogenix, Schiltigheim, France). An ε-amino palmitoylation of a lysine (K) residue was introduced in N-terminal position of each designed multi-
epitope peptide. These lipid tails were previously described to favor peptide presentation by antigen presenting cells (APC) [60]. Furthermore, it was reported that lipopeptides could constitute potent immunoadjuvants [61, 62]. In some designed multi-epitope peptides, the cysteines (S) of the original peptide sequence have been replaced by a glycine (G) or a serine (S) (underlined in the sequences) in order to avoid disulfide bridge formation, folding or multimerization of the peptide. To improve water-solubility, a hydrophobic spacer, as GR, was inserted between N-terminal lysine K and first epitope peptide [63].

The selected peptides were synthesized with more than 95% purity by Proteogenix (Schil-
tigheim, France).

Lyophilized peptides were dissolved according to their solubility in bi-distilled water, dimethyl sulfoxide (DMSO) or sodium bicarbonate, diluted in sterile double distilled water and stored at -80°C.

**Preparation of soluble *Leishmania* antigen (SLA)**

SLA was prepared from cultures of stationary phase *L. major* promastigotes (MHOM/TN/94/GLC94). Parasites were washed in 1× phosphate-buffered saline (PBS), centrifuged at 1000× g/10 min at 4°C and supernatants were removed. The pellets were resuspended in lysis buffer (50 mM Tris/5 mM EDTA/HCl, pH7.1 ml/ 1 × 10⁹ parasites), subjected to three rapid freeze/thaw cycles followed by three pulses of 20 s/40 W with sonicator. Samples were centrifuged at 5000× g for 20 min at 4°C, and supernatants were collected, aliquoted and stored at -80°C until use. Protein quantification was performed using Bradford method.

**Cell culture and stimulation**

Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood by density centrifugation through Ficoll-Hypaque (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Cells were cultured at a concentration of 10⁶/ml in RPMI 1640 medium supplemented with 100 IU/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine and 10% heat inactivated human AB serum (Sigma-Aldrich, St Louis, MO). Cells were plated in 96 well culture plates (Greiner Bio-One, Germany) and incubated overnight in a 5% CO₂ humidified atmosphere at 37°C for rest. Cells were then kept with media alone (unstimulated) or stimulated with either 1 µM of individual peptides or with peptide pools at 1µM/peptide. Recombinant human IL-2 (BD Bioscience, San Diego, CA) at 0.1 ng/ml final concentration was added after 24 hours and every 3 days along the culture period. Cell cultures were incubated for 10 days. As positive control cultures, cells were stimulated with Phytohemagglutinin (PHA) (Sigma-Aldrich, St Louis, MO) (10 µg/ml) and SLA (as indicator of previous disease) (10 µg/ml), for 5 days. Culture supernatants (in triplicate for each condition) were harvested and frozen at -80°C until use for ELISA and CBA assays, whereas cells were used for ELI-SPOT test and cell phenotyping.

**Enzyme Linked Immunosorbent Assay (ELISA)**

IFN-γ and IL-10 levels were measured in culture supernatants of PBMC exposed for 10 days to individual peptides or peptide pools or for 5 days to PHA or SLA. Human IFN-γ or IL-10 ELISA Sets (BD Biosciences, San Diego, CA) were used according to the manufacturer's instructions. Results were interpolated from a standard curve using recombinant cytokines and were expressed in pg/ml.
## Cytometric Bead Array assay (CBA)

TNF-α and GrB were quantified in culture supernatants of PBMC exposed for 10 days to peptide pools or 5 days to PHA or SLA, using the BD CBA Human Soluble Protein Flex Set system (BD Biosciences, San Diego, CA), according to the instructions of the manufacturer. In order to quantitate samples, the BD CBA Human Soluble Protein Flex Standard was performed for each cytokine and in each experiment. Data was acquired by flow cytometry (FACS Canto II, BD Bioscience, San Jose, CA). Flow Cytometric Analysis Program Array (FCAP Array; BD Biosciences) software was used for samples analysis.

## Intracellular cytokine staining and flow cytometry

After 10 days of *in vitro* stimulation with peptide pools, cells were washed and re-stimulated overnight with peptide pools (1 µM/peptide) and anti-CD49d/anti-CD28 antibodies (BD biosciences) as co-stimulators at 1 µg/ml final concentration. As positive control cultures, cells were stimulated with Phorbol Myristate Acetate (PMA) (50 ng/mL)/Ionomicyn (10⁻⁶ M) for 6 hours or with SLA (10 µg/ml) for 5 days. Cytokine secretion was stopped by Golgi stop (BD Biosciences, San Diego, CA) for the last 6 hours of culture. Cells were washed and incubated with anti-CD3-APC-H7 (SK7), anti-CD4-FITC (RPA-T4), anti-CD8-PE-Cy7 (RPA-T8), anti-CD45RO-APC (UCHL1) and anti-CCR7-PE (3D12) or with anti-CD3-APC-H7 (SK7), anti-CD4-FITC (RPA-T4), anti-CD8-PerCP-Cy5.5 (RPAT-8) or with anti-CD3-APC-H7 (SK7), anti-CD4-PerCP-Cy5.5 (RPA-T4) antibodies (BD biosciences, San Diego, CA), for 30 min at 4°C.

For intracellular staining, cells were washed, fixed and permeabilized using BD cytofix/Cytoperm plus kit (BD Biosciences, San Diego, CA) according to the manufacturer's instructions and co-stained with anti-IFN-γ-Alexa Fluor 647 (B27) and anti-GrB-PE (GB11) or with anti-IFN-γ-Alexa Fluor 647 (B27), anti-TNF-α-Alexa Fluor 488 (MAb11) and anti-IL-2-PE (MQ1-17H12) antibodies (BD biosciences, San Diego, CA), for 30 min at 4°C. Data acquisition was performed on a FACS canto II flow cytometer (BD Bioscience, San Jose, CA) and analysis was performed with FlowJo 7.6 software (TreeStar, Ashland OR). For acquisition, CompBeads Set Anti-Mouse Ig, k (Anti-Mouse Ig, k/Negative Control (FBS) Compensation Particles Set) (BD Biosciences, San Diego, CA), were used.

## Enzyme Linked Immunospot Assay (ELISpot)

ELISpot assay was performed with PBMC that were stimulated for 10 days with peptide pools (1µM/peptide), or for 5 days with SLA (10 µg/ml) or PHA (10 µg/ml) (positive controls), using the human interferon-γ ImmunoSpot kit (CTL, Germany), according to the manufacturer's recommendations. Briefly, 96-well PVDF filter plates were coated with human IFN-γ capture antibody. PBMCs (5.10⁵/ml) were washed, resuspended in CTL Test Medium and re-stimulated with peptide pools (1 µM/peptide) or with SLA (10 µg/ml) in the ELISPOT plates. PHA (10 µg/ml) and cells incubated with medium alone were used as positive and negative controls, respectively. Triplicate cultures were used. Cells were incubated for 24 hours in a 5% CO₂ humidified atmosphere at 37°C. Biotinylated anti-IFN-γ detection antibody was added and plates were incubated for 2 hours at room temperature. After washing, streptavidin-alkaline phosphatase conjugate was added for 30 min at room temperature. The spots were revealed by adding the substrate of the enzyme. Spots were counted using CTL ImmunoSpot reader (CTL Analyzers, ShakerHights, OH). Results were expressed as spot forming units (SFU) per 10⁶ PBMC.
**Statistical analysis**

Statistical analyses were performed with GraphPad Prism 5.0 (GraphPad Software, San Diego, CA). Results were expressed as medians (interquartile range, IQR as variances). Wilcoxon signed-rank test was used to compare median levels of cytokines between stimulated and unstimulated cultures or between percentages of cells producing cytokines after different stimulations. Mann-Whitney test was used for inter-groups analysis on normalized data after deducting the unstimulated value. p-values less than 0.05 were considered significant. Correlations were estimated using the Spearman rank (rs) correlation coefficient.

**Results**

*In silico* prediction of HLA-I restricted epitopes and design of multi-epitope peptides

We selected H2B, LmlRAB and PSA proteins as candidate antigens to map potential HLA-I restricted epitopes. In order to screen for conserved HLA-I restricted epitopes among *Leishmania* species, we first performed a protein sequence alignment from H2B and LmlRAB of *L. major* with corresponding protein sequences from *L. donovani*, *L. infantum*, *L. mexicana* and *L. braziliensis* species. The alignments showed 80.7 to 98.2% of identity for H2B and 63.6 to 90.8% of identity for LmlRAB. Protein sequence alignments from full *L. amazonensis* PSA sequence and PSA family from *L. infantum*, *L. donovani*, *L. tropica*, *L. chagasi* and *L. braziliensis*, showed 47.4 to 80.1% of identity. As we previously described, the central (LRR domain) and C-terminal regions of the PSA proteins are more divergent than the N-terminal region [64]. Predicted HLA-I restricted epitopes were selected from highly conserved regions of each protein or consensus sequence, according to (i) high binding scores from IEDB and NetMHC 3.2 servers (ii) promiscuity (epitopes presented by multiple HLA molecules from a supertype and, when it was possible, presented by multiple HLA-I supertypes), and (iii) low or no significant homology with human proteins. From these, a total of 17 epitopes were selected, among them 4, 6 and 7 HLA-I restricted epitopes were identified from H2B, LmlRAB and PSA, respectively (Table 3).

All the selected epitopes were predicted to have a strong or weak binding affinity for at least one of the HLA-I supertypes analyzed. To increase the coverage of the target population, we designed multi-epitopes peptides composed of HLA-I epitopes derived from each protein. We designed (i) one multi-epitope peptide containing four 9-mer HLA-I restricted epitopes derived from H2B (H2BI, 40-mer) (ii) two multi-epitope peptides, each containing three 9-mer HLA-I restricted epitopes derived from LmlRAB (RABI.2, 34-mer; RABI.3, 36-mer) and (iii) three multi-epitope peptides containing 8-, 9- or 10-mer HLA-I restricted epitopes derived from PSA (PSAI.3, 19-mer, 2 epitopes; PSAI.4, 18-mer, 2 epitopes and PSAI.5, 28-mer, 3 epitopes) (Patents WO/2014/102471, PCT/EP2019/064088) (Table 4).

Additional amino acids (K, KGR, KARY, ARY, RY or TV) were introduced to improve peptide proteasomal cleavage or water-solubility, or to introduce lipid tail in N-terminal position.

In summary, we have designed 6 HLA-I-restricted multi-epitope peptides, which collectively were predicted to bind with strong or weak affinity to all HLA-I supertypes, providing large population coverage (95.55% estimated population coverage) (Table 5).

*In silico* prediction of hot spot regions rich in HLA-II restricted epitopes and design of HLA-II-restricted multi-epitope peptides

Hot spots of promiscuous 15-mer HLA-II restricted epitopes were mapped from highly conserved regions or consensus sequences of H2B, LmlRAB and PSA proteins. Selected HLA-II
<table><caption>Table 3. Characteristics of *in silico* predicted HLA-I restricted epitopes derived from H2B, PSA and LmlRAB proteins.</caption><thead><tr><th>Protein</th><th>Main epitope sequence</th><th>HLA-I supertype SB<sup>a</sup> (IC50)<sup>a</sup></th><th>HLA-I supertype WB<sup>**</sup> (IC50)</th></tr></thead><tbody><tr><td rowspan="4">H2B</td><td>KAINAQMSM</td><td>HLA-B27 (42), HLA-B58 (16)</td><td>HLA-A01/03 (107), HLA-B62 (293)</td></tr><tr><td>MERICTEAA</td><td>HLA-B44 (24)</td><td></td></tr><tr><td>MSMSHRTMK</td><td>HLA-A01/A03 (41), HLA-A03 (5)</td><td>HLA-B27 (415)</td></tr><tr><td>SMSHRTMKI</td><td>HLA-A02 (10)</td><td>HLA-A01 (110), HLA-B08 (165), HLA-B27 (235)</td></tr><tr><td rowspan="8">PSA</td><td>TPEQRTNTL</td><td>HLA-B07 (18), HLA-B27 (50)</td><td>HLA-B08 (68)</td></tr><tr><td>ELGKKWIG</td><td>HLA-B08 (14)</td><td></td></tr><tr><td>TLPEMPVGV</td><td>HLA-A02 (2)</td><td></td></tr><tr><td>PEMPAGVDY</td><td></td><td>HLA-B44 (193)</td></tr><tr><td>ARGREGYFLA</td><td></td><td>HLA-B27 (233)</td></tr><tr><td>EGYFVTDEK</td><td>HLA-A03 (19)</td><td></td></tr><tr><td>GARGREGY</td><td></td><td>HLA-B58 (80)</td></tr><tr><td>LmlRAB</td><td>RFAQGEHDI</td><td></td><td>HLA-A24 (151)</td></tr><tr><td></td><td>RLPENAFVI</td><td>HLA-A01 (26), HLA-A02 (4)</td><td></td></tr><tr><td></td><td>TQGSSKAGF</td><td></td><td>HLA-B27 (120), HLA-B62 (73)</td></tr><tr><td></td><td>MPHVDQSSI</td><td>HLA-B07 (23)</td><td></td></tr><tr><td></td><td>ASFRSTEAI</td><td></td><td>HLA-A01 (103), HLA-B27 (62)</td></tr><tr><td></td><td>SSIMVVANK</td><td>HLA-A03 (7)</td><td>HLA-A01/A03 (94)</td></tr></tbody></table>

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restricted peptides were compared against *L. amazonensis*, *L. infantum*, *L. donovani*, *L. tropica*, *L. braziliensis*, *L. mexicana*, *L. chagasi* and *L. major* (sequence homologies ranging from 40.74 to 100%). Peptides were further selected based on low or no significant homology with human proteins.

One multi-epitope peptide derived from H2B (H2BII, 34-mer), two multi-epitope peptides derived from LmlRAB (RABII.4, 24-mer, RABII.5, 35-mer) and two multi-epitope peptides

<table><caption>Table 4. Characteristics of *in silico* predicted multi-epitope peptides.</caption><thead><tr><th>Protein</th><th>Peptide name</th><th>Synthetic multi-epitope sequence</th><th>AA<sup>*</sup> number</th><th>MW<sup>**</sup> (g/mol)</th><th>Peptide-binding HLA-I supertype concatenation</th></tr></thead><tbody><tr><td>H2B</td><td>H2BI</td><td>KARYMSMSHRTMKSMSHRTMKIKAINAQMSMMERICTEAA</td><td>40</td><td>4917.00</td><td>HLA-A01, HLA-A02, HLA-A01/A03, HLA-A03, HLA-B08, HLA-B27, HLA-B44, HLA-B58, HLA-B62</td></tr><tr><td rowspan="3">PSA</td><td>PSAI.3</td><td>KTPEQRTNTLTVELGKKWIG</td><td>19</td><td>2538.10</td><td>HLA-B07, HLA-B08, HLA-B27</td></tr><tr><td>PSAI.4</td><td>KTLP EmpVGVPEMPAGVDY</td><td>18</td><td>2268.83</td><td>HLA-A01, HLA-A02, HLA-A01/A24, HLA-B08, HLA-B44, HLA-B62</td></tr><tr><td>PSAI.5</td><td>KARGREGYFLARGREGYEGYFVTDEK</td><td>28</td><td>3579.13</td><td>HLA-A01, HLA-A02, HLA-A01/A03, HLA-A03, HLA-B27, HLA-B44, HLA-B58, HLA-B62</td></tr><tr><td rowspan="2">LmlRAB</td><td>RABI.2</td><td>KARYRFAQGEHDIRLPENAFVIARYTQGSSKAGF</td><td>34</td><td>4123.82</td><td>HLA-A01, HLA-A02, HLA-A24, HLA-B27, HLA-B62</td></tr><tr><td>RABI.3</td><td>KARYMPHVDQSSIARYASFRSTEAIRYSSIMVVANK</td><td>36</td><td>4373.20</td><td>HLA-A01, HLA-A01/A03, HLA-A03, HLA-B07, HLA-B27</td></tr></tbody></table>

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<footnote><sup>a</sup>IC50 values (nM) were predicted by NetMHC 3.2 and IEDB</footnote>

<footnote>*Strong binders (IC50<50nM).</footnote>

<footnote>**Weak binders (50nM>IC50<500nM)</footnote>

<footnote>*AA: Amino acid</footnote>

<footnote>**MW: Molecular weight (g/mol) obtained by mass-spectrometry</footnote>

<footnote>Letters in bold (K, KARY, ARY, RY, TV) represent spacers added to the multi-epitope sequence.</footnote>

<footnote>Underlined glycine (G) replaces a cysteine C</footnote>
<table><caption>Table 5. Estimated population coverage.</caption><thead><tr><th rowspan="2">Population/Area</th><th colspan="3">HLA-I</th><th colspan="3">HLA-II</th><th colspan="3">Combined HLA-I/-II</th></tr><tr><th>Coverage<sup>a</sup></th><th>Average_hit<sup>b</sup></th><th>pc90<sup>c</sup></th><th>Coverage<sup>a</sup></th><th>Average_hit<sup>b</sup></th><th>pc90<sup>c</sup></th><th>Coverage<sup>a</sup></th><th>Average_hit<sup>b</sup></th><th>pc90<sup>c</sup></th></tr></thead><tbody><tr><td>World</td><td>95.55%</td><td>4.51</td><td>1.81</td><td>99.59%</td><td>10.39</td><td>5.6</td><td>99.98%</td><td>14.91</td><td>9.2</td></tr></tbody></table>

<sup>a</sup>Projected population coverage

<sup>b</sup>Average number of epitope hits / HLA combinations recognized by the population

<sup>c</sup>Minimum number of epitope hits / HLA combinations recognized by 90% of the population

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derived from PSA (PSAII.6, 29-mer, PSAII.7, 31-mer) (Patent PCT/EP2019/064088), were designed (Table 6).

Additional amino acids (K or KGR) were introduced to improve peptide water-solubility or to introduce lipid tail in N-terminal position. Peptides rich in HLA-II epitopes were predicted to bind with strong or weak affinity to the most frequent 18 HLA-II alleles, representative of HLA-II supertypes. In total, we have designed 5 HLA-II restricted multi-epitope peptides with strong or weak affinity to all HLA-II supertypes, providing wide population coverage estimated to 99.59% (Table 5). It should be noted that the designed multi-epitope peptides, having a high affinity for several HLA-I and -II molecules, if taken all together as vaccine candidate components, could have a strong immunoprevalent potential in leishmaniasis endemic areas (Table 5).

## Cytokine responses to multi-epitope peptides in cured CL subjects

We evaluated the ability of the multi-epitope peptides, used either individually (N = 11) or as peptide pools (N = 25), to induce IFN-γ and IL-10, in individuals with cured CL and in healthy subjects, by ELISA.

Peptides pools have been constituted to group HLA-I, HLA-II or HLA-I and HLA-II peptides, derived from the same or different proteins (Table 7).

As expected, high significant levels of IFN-γ were observed in cured CL, but not in healthy individuals, in response to SLA stimulation (p<0.0001) (Fig 1A and 1B).

No significant differences in IFN-γ induction were observed between stimulated and unstimulated cultures, after stimulation of PBMC from ten cured CL individuals with multi-epitope peptides used individually (Fig 1A). However, among these peptides, H2BI and H2BII showed the highest IFN-γ levels, although not statistically significant when compared to unstimulated cultures. We further analyzed these 2 peptides in a larger group of healed CL and in healthy individuals. As shown in Fig 1, significant levels of IFN-γ were detected in healed CL, when compared to unstimulated cultures (Fig 1B). In addition, H2BI and H2BII were not able to induce a significant IFN-γ response in healthy individuals (Fig 1B).

Significant levels of IFN-γ were observed in response to two HLA-I (P1 and P4) (Fig 2A), four HLA-II (P5, P6, P7 and P8) (Fig 2B) and ten HLA-I/HLA-II peptide pools (P9, P12, P13, P14, P15, P16, P17, P18, P19 and P21) (Fig 3).

These responses were specific as no significant IFN-γ levels were detected in healthy individuals, except for P12 (Figs 2 and 3). This peptide pool induced significant levels of IFN-γ in cured CL as well as in the healthy group, suggesting that this response was not a feature of *Leishmania* infection. Interestingly, IFN-γ levels induced by P21 pool (including all multi-epitope peptides) in cured CL individuals (median/IQR: 1207/928) were similar to those induced by SLA in the same group (1182/1837), (p>0.05) (Fig 3).

No IL-10 was observed in response to peptides used either individually (except low but significant levels observed for H2BII) or as peptide pools (Figs 4 and 5).
<table><caption>Table 6. Characteristics of *in silico* predicted sequence rich in HLA-II-restricted epitopes derived from H2B, PSA and LmlRAB proteins.</caption><thead><tr><th>Protein</th><th>Peptide name</th><th>Sequence rich in HLA-II epitopes</th><th>AA number</th><th>MW (g/mol)</th><th>HLA-II supertype SB<sup>*</sup>(IC50)<sup>a</sup></th><th>HLA-II supertype WB<sup>**</sup>(IC50)</th></tr></thead><tbody><tr><td>H2B</td><td>H2BII</td><td>KGRKPKRSWNVYVGRSLKAINAQMSMSHRTMKIV</td><td>34</td><td>4199.23</td><td>DR1: DRB1*0101 (4), DR7: DRB10701 (19), DR9: DRB1*0901 (43), DR11/DR12: DRB1*1101/DRB1*1201 (44), DRB5: DRB5*0101 (43), DRB4: DRB4*0101 (13)</td><td>DR15:DRB1*1501 (62), DR4: DRB1*0401 (86), DR4: DRB1*0405 (87), DR4: DRB1*0802 (127), DR3: DRB1*0301 (64), DR13: DRB1*1302 (300), DP1: DPB1*0101 (313), DP402: DPB1*402 (415)</td></tr><tr><td rowspan="2">PSA</td><td>PSAII.6</td><td>KGRAARSARSGEGYFLTDEKTSLVYGDGG</td><td>29</td><td>3287.76</td><td>DR3: DRB1*0301 (8), DRB3: DRB3*0101/DRB3*0202 (26)</td><td>DR1: DRB1*0101 (123), DR7: DRB10701 (342), DR11/DR12: DRB1*1101/DRB1*1201 (156), DR4: DRB1*0401 (70), DP1: DPB1*0101 (302), DP402: DPB1*402 (382)</td></tr><tr><td>PSAII.7</td><td>KGREMPSGSDYSHSMIRDLDFSNMGLLLSGT</td><td>31</td><td>3641.28</td><td>DR1: DRB1*0101 (39), DR7: DRB10701 (14), DR4: DRB1*0401 (22)</td><td>DR9: DRB1*0901 (65), DR11/ DR12: DRB1*1101/DRB1*1201 (275), DR15: DRB1*1501 (329), DRB5: DRB5*0101 (215), DR4: DRB1*0405 (64), DR4: DRB1*0802 (317), DR3: DRB1*0301 (263), DR13: DRB1*1302 (197), DRB4 (146), DRB3: DRB3*0101/DRB3*0202 (130), DP1: DPB1*0101 (98), DP402: DPB1*402 (30), DP2: DPB1*0201 (374), DP401: DPB1*0401 (224)</td></tr><tr><td rowspan="2">LmlRAB</td><td>RABII.4</td><td>KGRLHSVVGRHLSIVADHMPHLDQ</td><td>24</td><td>2941.58</td><td>DR1: DRB1*0101 (13), DR7: DRB10701 (6), DR13: DRB1*1302 (27), DRB3: DRB3*0101/DRB3*0202 (16), DRB4: DRB4*0101 (48)</td><td>DR9: DRB1*0901 (424), DR11/ DR12: DRB1*1101/DRB1*1201 (86), DR15: DRB1*1501 (74), DRB5: DRB5*0101 (177), DR4: DRB1*0401 (67), DR4: DRB1*0405 (196), DR4: DRB1*0802 (292), DR3: DRB1*0301 (133)</td></tr><tr><td>RABII.5</td><td>KGRSENAIVTSRKVQEEVFDLFTDVHSEVSAKTK</td><td>35</td><td>4237.91</td><td>DR7: DRB1*10701 (31), DR11/ DR12: DRB1*1101/ DRB1*1201 (32), DR3: DRB1*0301 (38), DP1: DPB1*0101 (34), DP2: DPB1*0201 (11), DP401: DPB1*0101 (32)</td><td>DR1: DRB1*0901 (286), DR9: DRB1*0901 (400), DR15: DRB1*1501 (141), DRB5: DRB5*0101 (59), DR4: DRB1*0401 (63), DR4: DRB1*0405 (272), DR4: DRB1*0802 (199), DRB3: DRB3*0101/DRB3*0202 (274) DP402: DPB1*402 (259), DP5: BPB1*0501 (84)</td></tr></tbody></table>

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However, a suppressive effect on IL-10 production was observed after stimulation with PII, P6, P8, P10, P13, P14 and P18 (Figs 4C and 5).

We further analyzed TNF-α and GrB, by CBA, in response to P21 pool, in culture supernatants from PBMC issued from cured CL and healthy individuals (Fig 6).

Significant TNF-α levels were induced by P21 in cured (median/IQR in stimulated; unstimulated cultures: 10/13; 0/1.9) (p = 0.001), but not in healthy individuals (5.4/2; 1.9/10)

<footnote><sup>a</sup> IC50 values (nM) were predicted by NetMHCII 2.2 and IEDB.</footnote>

<footnote>*Strong binders.</footnote>

<footnote>**Weak binders. Underlined serine (S) replaces a cysteine C.</footnote>
<table><caption>Table 7. Peptide pools composition.</caption><thead><tr><th>Protein</th><th colspan="2">H2B</th><th colspan="5">PSA</th><th colspan="4">RABGTPase</th></tr><tr><th>Peptides</th><th>H2BI</th><th>H2BII</th><th>PSAI.3</th><th>PSAI.4</th><th>PSAI.5</th><th>PSAI.6</th><th>PSAI.7</th><th>RABI.2</th><th>RABI.3</th><th>RABII.4</th><th>RABII.5</th></tr></thead><tbody><tr><td colspan="12">Pools including HLA-I peptides</td></tr><tr><td>PI</td><td></td><td></td><td>x</td><td>x</td><td>x</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>RI</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>x</td><td>x</td><td></td><td></td></tr><tr><td>P1</td><td>x</td><td></td><td>x</td><td>x</td><td>x</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>P2</td><td>x</td><td></td><td></td><td></td><td></td><td></td><td></td><td>x</td><td>x</td><td></td><td></td></tr><tr><td>P3</td><td></td><td></td><td>x</td><td>x</td><td>x</td><td></td><td></td><td>x</td><td>x</td><td></td><td></td></tr><tr><td>P4</td><td>x</td><td></td><td>x</td><td>x</td><td>x</td><td></td><td></td><td>x</td><td>x</td><td></td><td></td></tr><tr><td colspan="12">Pools including HLA-II peptides</td></tr><tr><td>PII</td><td></td><td></td><td></td><td></td><td></td><td>x</td><td>x</td><td></td><td></td><td></td><td></td></tr><tr><td>RII</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>x</td><td>x</td><td></td></tr><tr><td>P5</td><td></td><td>x</td><td></td><td></td><td></td><td>x</td><td>x</td><td></td><td></td><td></td><td></td></tr><tr><td>P6</td><td></td><td>x</td><td></td><td></td><td></td><td></td><td></td><td></td><td>x</td><td>x</td><td></td></tr><tr><td>P7</td><td></td><td></td><td></td><td></td><td></td><td>x</td><td>x</td><td></td><td>x</td><td>x</td><td></td></tr><tr><td>P8</td><td></td><td>x</td><td></td><td></td><td></td><td>x</td><td>x</td><td></td><td>x</td><td>x</td><td></td></tr><tr><td colspan="12">Pools including HLA-I and HLA-II peptides</td></tr><tr><td>P9</td><td>x</td><td>x</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>P10</td><td></td><td></td><td>x</td><td>x</td><td>x</td><td>x</td><td>x</td><td></td><td></td><td></td><td></td></tr><tr><td>P11</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>x</td><td>x</td><td>x</td><td>x</td></tr><tr><td>P12</td><td>x</td><td></td><td></td><td></td><td>x</td><td>x</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>P13</td><td>x</td><td>x</td><td></td><td></td><td>x</td><td>x</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>P14</td><td>x</td><td></td><td></td><td></td><td>x</td><td>x</td><td></td><td>x</td><td>x</td><td></td><td></td></tr><tr><td>P15</td><td></td><td>x</td><td></td><td>x</td><td>x</td><td></td><td></td><td></td><td></td><td>x</td><td>x</td></tr><tr><td>P16</td><td>x</td><td>x</td><td>x</td><td>x</td><td>x</td><td>x</td><td>x</td><td></td><td></td><td></td><td></td></tr><tr><td>P17</td><td>x</td><td>x</td><td></td><td></td><td></td><td></td><td></td><td>x</td><td>x</td><td>x</td><td>x</td></tr><tr><td>P18</td><td>x</td><td></td><td></td><td>x</td><td>x</td><td></td><td></td><td></td><td></td><td>x</td><td>x</td></tr><tr><td>P19</td><td></td><td>x</td><td></td><td></td><td></td><td>x</td><td>x</td><td>x</td><td>x</td><td></td><td></td></tr><tr><td>P20</td><td></td><td></td><td>x</td><td>x</td><td>x</td><td>x</td><td>x</td><td>x</td><td>x</td><td>x</td><td>x</td></tr><tr><td>P21</td><td>x</td><td>x</td><td>x</td><td>x</td><td>x</td><td>x</td><td>x</td><td>x</td><td>x</td><td>x</td><td>x</td></tr></tbody></table>

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<figure><img src="image_4.png" /><figcaption>**Fig 1. IFN-γ responses to multi-epitope peptides.** IFN-γ was quantified by ELISA, in the culture supernatants of PBMC from cured CL subjects (N = 10) stimulated with multi-epitope peptides (1 µM), for 10 days (A). PHA and SLA (10 µg/ml) were used as positive controls. Unstimulated cultures (medium) were used as negative controls. H2BI and H2BII peptides were further evaluated in a larger number of cured CL (N = 40) as well as in healthy individuals (N = 12) (B). Horizontal bars represent median values. Statistical significance was assigned to a value of p<0,05 (**p<0.01, ***p<0.001).</figcaption></figure>

https://doi.org/10.1371/journal.pntd.0008093.g001
<figure><img src="image_5.png" /><figcaption>**Fig 2.** IFN-γ responses to pools composed of HLA-I or HLA-II peptides. IFN-γ was quantified by ELISA, in culture supernatants of PBMC from cured CL subjects (N = 23) or healthy individuals (N = 12) stimulated with peptide pools (1 µM/peptide) composed of HLA-I (A) or HLA-II (B) peptides, for 10 days. PHA and SLA were used as positive controls (10 µg/ml). Horizontal bars represent median values. Statistical significance was assigned to a value of p<0.05 (*p*<0.05, **p**<0.01, ***p**<0.001).</figcaption></figure>

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(p>0.05) (Fig 6A). No GrB response was observed after P21 stimulation in cured CL individuals (Fig 6B). High and significant levels of TNF-α and GrB were observed in cured CL but not in healthy individuals, in response to SLA stimulation (p<0.005) (Fig 6).

For further analysis, and because PBMC numbers was a limiting factor, we selected peptide
pools inducing the highest significant and specific IFN-γ levels. These pools included P21
(median/IQR in stimulated; unstimulated cultures: 1207/928; 15/93), P16 (1080/1249; 12/53),
P13 (946/1229; 12/65), P14 (730/1052; 12/65), P9 (584/1148; 33/64), and P8 (287/1177; 15/64)
(p <0.0003). Among these pools, P21, P16 and P13, sharing H2BI, H2BII and PII, induced the
highest IFN-γ levels, suggesting that the combination of these peptides would be sufficient to
induce IFN-γ levels as high as those induced by the P21 pool that contains 11 peptides.
<figure><img src="image_6.png" /><figcaption>**Fig 3.** IFN-γ responses to pools composed of both HLA-I and HLA-II peptides. IFN-γ was quantified by ELISA, in culture supernatants of PBMC from cured CL subjects (N = 23) or healthy individuals (N = 12) stimulated with peptide pools (1 µM/peptide) composed of both HLA-I and HLA-II peptides, for 10 days. PHA and SLA were used as positive controls (10 µg/ml). Horizontal bars represent median values. Statistical significance was assigned to a value of p<0.05 (*p<0.05, **p<0.01, ***p<0.001).</figcaption></figure>

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## Quantification of specific IFN-γ producing T lymphocytes by ELISpot assay

The ability of multi-epitope peptide pools to activate *Leishmania*-reactive CD4+ T cells producing IFN-γ was assessed by ELISpot assay. Results were expressed as SFU per 10⁶ PBMC and compared between stimulated and unstimulated cultures. We showed significant responses against peptide pools P8 (median/IQR in stimulated; unstimulated cultures: 857/893; 8/24), P9 (95/1392; 10/27), P14 (135/125; 20/31), P16 (550/1933; 8/22) and P21 (542/762; 10/27) (p≤0.03) (Fig 7).

Interestingly, when compared to SLA induced response; no statistically significant difference was observed for P8, P9, P14, P16 and P21 pools. The IFN-γ response induced by P13 was not significantly increased when compared to unstimulated culture. This peptide pool could only be analyzed in 5 individuals and may need further analysis in a larger group of individuals to validate this negative result.

## HLA-typing and IFN-γ response

HLA typing was performed for five cured CL individuals. IFN-γ responses to P8, P9, P16 and P21 stimulation, were analyzed by ELISPOT (S1 Table). SFU/10⁶PBMC were considered positive if above the threshold defined as mean values observed in unstimulated cultures +2SD. A positive IFN-γ response was observed in response to P8, P9, P16 and P21 in four among five cured CL individuals (S1 Table). In contrast, no IFN-γ response was detected in CCL64 individual, after stimulation with P9 composed of H2BI and H2BII peptides. This donor does not express HLA-I alleles to which H2BI epitopes are able to bind, but carries HLA-II alleles for which H2BII epitopes have a strong affinity. The positive response observed to P8, P16 and P21, in this individual, could be induced by HLA-II-restricted epitopes derived from PSA protein shared by these peptide pools.

## Frequencies of specific cytokine secreting T cells after multi-epitope peptide stimulation

PBMC stimulated by the selected peptide pools were stained and analyzed by flow cytometry to first determine the percentage of IFN-γ or GrB-producing CD4+ T cells among total CD4+ T cells, in cured CL subjects. We observed a significantly higher percentage of CD4+T cells
<figure><img src="image_7.png" /></figure>
**Fig 4. IL-10 responses to multi-epitope peptides used individually or as pools composed of HLA-I or HLA-II peptides.** IL-10 was quantified by ELISA, in culture supernatants of PBMC from cured CL subjects (N = 16) or healthy individuals (N = 10) stimulated with multi-epitope peptides (1 µM) used individually (A), or as peptide pools composed of HLA-I (B) or HLA-II (C) peptides, for 10 days. PHA and SLA were used as positive controls (10 µg/ml). Horizontal bars represent median values. Statistical significance was assigned to a value of p<0.05 (*p<0.05, **p<0.01, ***p<0.001).

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producing IFN-γ in response to all selected peptide pools, in comparison to unstimulated cultures (median/IQR in stimulated; unstimulated cultures: P8: 0.7/1.9; 0.2/0.7, P9: 1.6/2.5; 0.3/0.6, P13: 4/6; 0.1/0.6, P14: 1.1/1.1; 0.2/0.7, P16: 3/4; 1/1.3 and P21: 1.2/3.1; 0.1/0.6) (p≤0.009) (Fig 8A and S1 Fig).

With regard to CD4+ T cells producing GrB, we showed a significant increase in their percentages after stimulation with P8 (4/12; 1.4/3.6), P9 (3.7/6.1; 1.4/3.6), P16 (4/11; 1.4/3.6) and P21 (6/12; 1.4/3.6) (p≤0.03) (Fig 8B and S1 Fig). The response induced by P13 peptide pool (10/18; 2/3) was not statistically significant. This result may need to be further studied in a larger group of individuals in order to be validated. We also assessed IFN-γ+/GrB+ CD4+ T cells among total CD4+ T cells and we showed a significant increase for this population after P21 stimulation (1.7/3.1; 0.1/0.5) (p = 0.01) (S2 Fig). No correlations were observed between IFN-γ+ CD4+ T cells and GrB+ CD4+ T cells.

We next assessed intracellular IFN-γ production by peptide-specific CD8+ T cells in cured CL individuals (Fig 9 and S3 Fig).

Results were expressed as percentages of IFN-γ+ CD8+ T cells among total CD8+ T cells and were compared between stimulated and unstimulated cultures. As shown in Fig 9, a significant increase in the percentage of IFN-γ+ CD8+ T cells was observed after stimulation with P8 (median/IQR in stimulated; unstimulated cultures: 2.5/2.6; 2/1.4), P9 (2.6/2.8; 2/1.4), P13 (6.1/3.8; 2.1/0.9), P16 (3.3/4.8; 2/1.4) and P21 (4.6/7; 2/1.4) (p≤0.03).

Finally we evaluated the ability of peptide pools to stimulate a multifunctional CD4+ T cell response, using Boolean gating (S4 Fig). As shown in Fig 10, no significant increase was observed in the percentage of CD4+ T cells producing IFN-γ, IL-2 and TNF-α, in response to both peptide pools and SLA in cured CL individuals.

<figure><img src="image_8.png" /></figure>

**Fig 5. IL-10 responses to pools composed of both HLA-I and HLA-II peptides.** IL-10 was quantified by ELISA, in culture supernatants of PBMC from cured CL subjects (N = 16) or healthy individuals (N = 10) stimulated with peptide pools (1µM/peptide) composed of both HLA-I and HLA-II peptides, for 10 days. PHA and SLA were used as positive controls (10 µg/ml). Horizontal bars represent median values. Statistical significance was assigned to a value of p<0.05 (*p<0.05, **p<0.01, ***p<0.001).

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<figure><img src="image_9.png" /><figcaption>**Fig 6. TNF-α and Granzyme B responses to P21 peptide pool.** TNF-α (A) and GrB (B) were quantified by CBA, in the culture supernatants of PBMC from cured subjects (N = 11) or healthy individuals (N = 7) stimulated with P21 (1 µM), for 10 days. PHA and SLA (10 µg/ml) were used as positive controls. Horizontal bars indicate median values. Statistical significance was assigned to a value of p<0.05 (*(p<0.05, **p<0.01).</figcaption></figure>

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We also investigated whether these T cells display a monofunctional or bifunctional profile in response to peptide stimulation. A significant increase in the frequencies of bifunctional IFN-γ+/TNF-α+, IL-2+/TNF-α+, and monofunctional IFN-γ+ and TNF-α+ CD4+ T cells, was shown in response to SLA and to peptide pools P13, P14 and P21 (p<0.04) (Fig 10). P9 pool was also able to induce high significant percentages of IFN-γ+/TNF-α+, IFN-γ+ and TNF-α+ CD4+ T cells (Fig 10). Surprisingly, we did not detect any changes in these populations in response to P16 pool, probably due to the low number of individuals analyzed. P21 was the only pool to induce a significant increase in the IL-2+ CD4+ T cell population.

## Frequencies of specific memory T cells after multi-epitope peptide stimulation

Phenotypic analysis of memory T cells in response to the selected multi-epitope peptide pools (N = 6) was performed in cured CL individuals (Fig 11 and S5 Fig).
<figure><img src="image_10.png" /><figcaption>**Fig 7. Enumeration of IFN-γ-secreting T cells in response to selected multi-epitope peptide pools.** PBMC from cured CL subjects (N = 11), previously stimulated with selected peptide pools (1 µM/peptide) for 10 days, were re-stimulated for 24 h with the same peptide pools at the same concentration. PHA and SLA were used as positive controls (10 µg/ml). Results are expressed as spot forming units (SFU) per million PBMCs. Horizontal bars represent median values. Statistical significance was assigned to a value of p<0.05 (*p<0.05, **p<0.01, ***p<0.001).</figcaption></figure>

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We used CD45RO and CCR7 to distinguish T memory T cells into TCM (CD45RO+CCR7+) or TEM (CD45RO+CCR7-). Frequencies of peptide-specific TCM and TEM were assessed among total CD4+ or CD8+ T cell subsets. A significant increase in the percentage of CD4+ TCM as well as CD8+ TCM cells was observed in response to P8 (median/IQR in stimulated; unstimulated cultures for CD4+ and CD8+ TCM: 1.1/1.3; 0.3/0.1% and 0.3/0.3; 0.06/0.06), P13 (2/2; 0.4/0.8% and 0.5/1.3; 0.06/0.2), P14 (2/0.9; 0.3/0.1% and 0.3/0.4; 0.06/0.06) and P21 (2.3/1.4; 0.4/0.8% and 0.9/1.3; 0.06/0.2) (p≤0.04) (Fig 11A and 11C).

There was no significant change in the percentage of CD4+ TEM cells after peptide stimulation (Fig 11B).

The cured CL individuals exhibited high frequencies of both CD4+ TCM and TEM (median/IQR in stimulated; unstimulated cultures: 0.8/2; 0.3/0.9 and 59/15; 44/24) and CD8+ TCM and TEM cells (0.2/0.8; 0.05/0.2 and 43/28; 29/23), after SLA stimulation (p≤0.002) (Fig 11).

## Discussion

Vaccination is the best option to induce a successful host protective immunity against *Leishmania* parasites. There is currently no licensed vaccine for human use despite strong arguments supporting its feasibility. Peptide-based vaccines are composed by the most relevant synthetic short peptide fragments, making them safe, stable and easy to produce [40, 65–68].

Suitable human peptide-based vaccine candidates should contain both HLA-I and -II restricted epitopes, able to generate a properly balanced T cell response, since both CD4+ and CD8+ T cells are critical for mediating protective immune responses against *Leishmania* parasites.

In this study, we have selected three *Leishmania* proteins; H2B, PSA and LmlRAB for the prediction of HLA-I and -II binding peptides, using online available algorithms. These proteins were able to confer significant protection in mice and dogs [47–52, 69, 70]. However, these results should be interpreted with caution since the protection described in these animal models, which was induced against needle challenge with parasites, does not necessarily translate to protection following challenge by infected sand flies. Vaccine failure against infected sand fly challenge was associated with the inability of the vaccine to replicate the early kinetics of the secondary immunity observed in animals with a healed primary infection [71–73].
<figure><img src="image_11.png" /><figcaption>**Fig 8.** Percentages of CD4+ T cells producing IFN-γ or GrB in response to selected multi-epitope peptide pools.
PBMC from cured CL subjects, previously stimulated with selected peptide pools (1 µM/peptide) for 10 days, were re-stimulated overnight with the same peptide pools (1 µM/peptide) and with anti-CD49d/anti-CD28 antibodies (1 µg/ml). For intracellular IFN-γ (N = 21 cured individuals) and Granzyme B (N = 9 cured individuals) detection, cells were treated with GolgiStop for 6 h of culture, fixed and permeabilized using BD Cytoperm/cytofix kit. Data were analyzed by FlowJo software. PBMC stimulated with PMA (50 ng/ml)/Ionomicin (10⁻⁶M) for 6 h or SLA (10µg/ml) for 5 days, were used as positive control. Results represent the frequency of CD4+ IFN-γ+ (A) and CD4+ granzyme B+ (B) T cell populations. Horizontal bars represent median values. Statistical significance was assigned to a value of p<0.05 (*p*<0.05, **p**<0.01, ***p**<0.001).</figcaption></figure>

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Nervertheless, we have previously reported that *Leishmania* H2B [49], *L. amazonensis* PSA protein (LaPSA-38S) [47] and LmlRAB [48], were able to induce a dominant Th1 profile of immune response in cured CL individuals, suggesting that these proteins are potential vaccine candidates against human leishmaniasis.

H2B, PSA and LmlRAB proteins were used to design eleven multi-epitope peptides composed of highly scored HLA-I or -II restricted epitopes, which were predicted to bind to at least 3/12 of the most common HLA-I and 3/5 of the most common HLA-II supertypes. It is interesting to note that, in combination, these multi-epitope peptides theoretically cover all HLA-I and -II supertypes analyzed, thus providing an optimal coverage in the human population. All peptides share a high to moderate level of sequence homology with different *Leishmania* species, a requisite for cross-species protection.
<figure><img src="image_12.png" /><figcaption>**Fig 9.** Percentages of CD8+ T cells producing IFN-γ in response to selected multi-epitope peptide pools. PBMC from cured CL subjects (N = 9), previously stimulated with selected peptide pools (1 µM/peptide) for 10 days, were re-stimulated overnight with the same peptide pools (1 µM/peptide) and with anti-CD49d/anti-CD28 antibodies (1 µg/ml). For intracellular IFN-γ detection, cells were treated with Golgestop for 6 h of culture, fixed and permeabilized using BD Cytoperm/cytofix kit. Data were analyzed by FlowJo software. PBMC stimulated with PMA (50 ng/ml)/Ionomicyn (10⁻⁶M) for 6 h or SLA (10 µg/ml) for 5 days, were used as positive control. Results represent the frequency of CD8+ IFN-γ+ T cell populations. Horizontal bars represent median values. Statistical significance was assigned to a value of *p*<0.05 (*p*<0.05, **p*<0.01).</figcaption></figure>

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Most of the studies have focused on the identification of HLA-I epitopes [74–80] but only a few have been conducted to predict HLA-II [81–83] or a combination of both HLA-I and -II epitopes [23, 84, 85]. In this study, we described peptides containing multiple HLA-I and -II epitopes that would induce both CD8+ and CD4+ T cell responses, crucial for protection.

To confirm the ability of the predicted peptides to induce a specific immune response, we evaluated their immunogenicity in PBMC from individuals with healed CL due to *L. major*. It is well established that IFN-γ is a key effector cytokine, crucial to eliminate *Leishmania* parasites. Elevated levels of this cytokine are observed after *in vitro* stimulation, with *Leishmania* antigens, of PBMC from individuals who have developed a protective immunity against *L. major* [11, 86, 87]. We showed significant IFN-γ levels in response to pools containing HLA-I, HLA-II or HLA-I and -II multi-epitope peptides in cured CL individuals, whereas we could not detect any significant IFN-γ production in healthy subjects, indicating the *Leishmania* specificity of the responses observed. Furthermore, we noticed that most of the peptide pools containing both HLA-I and -II epitopes induced higher amounts of IFN-γ than those containing only HLA-I or -II epitopes. These results emphasize the importance to include both CD4+ and CD8+ specific T cell epitopes in peptide based vaccines [67, 88, 89].

Significant and specific levels of TNF-α, a cofactor for macrophage activation, were also observed in response to P21 peptide pool, in cured individuals. With regard to IL-10 production, we did not detect significant production in response to peptides used either individually or combined. Rather, some peptide pools induced a suppressive effect on IL-10 production. This anti-inflammatory cytokine has been involved in parasite persistence and disease establishment but also in controlling an excessive inflammatory response [15, 18, 90]. The immunomodulatory effect of some of our peptides may not be beneficial to the host, given the role of IL-10 in the regulation of excessive IFN-γ responses [18]. A suppressive effect has also been reported in response to peptides derived from KMP-11 or GP63 proteins [40, 81].
<figure><img src="image_13.png" /><figcaption>**Fig 10.** Multifunctional CD4+ T cell responses after multi-epitope peptide pools stimulation. PBMC from cured CL subjects (5 to 15 individuals), previously stimulated with selected peptide pools (1 µM/peptide) for 10 days were re-stimulated overnight with the same peptide pools (1 µM/peptide) and with anti-CD49d/ anti-CD28 antibodies (1 µg/ml). For intracellular cytokine detection, cells were treated with GolgiStop for 6 h of culture, fixed and permeabilized using BD Cytoperm/cytofix kit. Data were analyzed by FlowJo software. PBMC stimulated with PMA (50 ng/ml)/Ionomicin (10⁻⁶M) for 6 h or SLA (10µg/ml) for 5 days, were used as positive control. A five-color flow cytometry panel was used to simultaneously analyze multiple cytokine at the single cell level. Boolean combinations of the three cytokine gates (IFN-γ, IL-2 and TNF-α) were used to uniquely discriminate responding cells based on their functionality. The frequency of CD4+ T cells expressing each of the seven possible combinations of IFN-γ, IL-2 and TNF-α was determined. Horizontal bars represent median values. Statistical significance was assigned to a value of p<0.05 (*p<0.05, **p<0.01, ***p<0.001).</figcaption></figure>

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These results suggest that multi-epitopes used as pools, can elicit a Th1-biased response, in individuals with healed CL. It is interesting to note that, among multi-epitope peptides used individually, H2BI and H2BII were able to induce significant levels of IFN-γ in cured CL individuals. In addition, all peptide pools, that were able to induce specific and significant IFN-γ, a part from P7, share HLA-I and/or HLA-II peptides derived from the H2B protein.

Some studies have shown significant levels of IFN-γ and low levels of IL-10 in response to HLA-I or HLA-II restricted epitopes derived from different *Leishmania* proteins, used alone or as pools, in cured CL or visceral leishmaniasis (VL) individuals [75, 77, 78, 83]. However, only a few studies have focused on the identification of both HLA-I and -II restricted peptides. Similar to our results, both highly promiscuous HLA-DR and HLA-I-binding epitopes, identified within nucleoside hydrolase NH36 domains, have been shown to induce IFN-γ production, though low, in asymptomatic DTH+ subjects [23]. Another study identified T cell epitopes with high affinity for both human HLA-I and -II within the proteome of L.
<figure><img src="image_14.png" /><figcaption>**Fig 11.** Phenotyping of multi-epitope peptide-specific CD4+ and CD8+ memory T cells. PBMC from cured CL subjects (N = 11), previously stimulated with selected peptide pools (1 µM/peptide) for 10 days, were re-stimulated overnight with the same peptide pools (1 µM/peptide) and with anti-CD49d/anti-CD28 antibodies (1 µg/ml). The percentages of CD45RO+CCR7+CD4+ or CD8+ TCM (A, C) and, CD45RO+CCR7-CD4+ or CD8+ TEM (B, D), were analyzed by FlowJo software. PBMC stimulated with SLA (10 µg/ml) for 5 days, were used as positive control. Statistical significance was assigned to a value of p<0,05 (*p<0.05, **p<0.01, ***p<0.001).</figcaption></figure>

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*braziliensis*, which were able to stimulate the proliferation of lymphocytes from healed CL individuals, although the cytokine production was not evaluated [84]. Peptides including both highly scored murine MHC-I and II restricted epitopes derived from *L. infantum* proteins, induced high amounts of IFN-γ and low IL-10 levels in spleen cell culture supernatants from immunized mice [40]. More recently, it has been reported that recombinant chimeric proteins composed by human and murine MHC-I and II-specific T cell epitopes, were able to induce high IFN-γ/IL-10 ratios in both immunized mice and treated VL subjects [85, 91]. In addition, these recombinant chimeric proteins induced a protective immune response in vaccinated mice [85, 91]. On the other hand, some authors have identified HLA-I or -II-binding peptides that were unable to induce IFN-γ production in healed CL or VL subjects [76, 81]. The fact that synthetic epitopes alone are not immunogenic enough and the use of different culture conditions may explain these different results.

The reason we used multi-epitope peptides in our work is that these peptides are longer than isolated epitopes and would therefore be more immunogenic. Furthermore, a palmitoylated tail was added to our peptides, probably improving their immunogenicity [60, 61].

For further analysis, six peptide pools inducing the highest IFN-γ levels were selected. We validated their ability to activate specific IFN-γ producing cells in healed CL individuals, by ELISpot assay.

HLA typing was performed for some volunteers, for whom IFN-γ positive responses were shown in response to peptide pools, thus validating the use of *in silico* methods for predicting epitopes in our proteins. However, a negative response was observed for one individual,
probably due to the absence of HLA to which epitopes are able to bind or the absence of the predicted epitopes during natural infection. As previously reported for other antigens, not all of HLA binders lead to T cell stimulation [75, 92, 93].

Both CD4+ and CD8+ IFN-γ-secreting T cells responses are mandatory for *Leishmania* parasites elimination [10, 24, 26, 32, 36, 94]. An ideal vaccine candidate should therefore be able to induce both CD4+ and CD8+ T cell responses. We demonstrated that peptide pools were able to induce a significant increase of the percentages of both IFN-γ-producing CD4+ and CD8+ T cells (except P14), in cured individuals. These results suggest that the selected peptides include T cell epitopes that are naturally processed and presented to the immune system during infection. Some studies have shown that HLA-I or HLA-II restricted T cell epitopes derived from *L. major* or *L. donovani* proteins, used alone or as pools, were able to activate specific IFN-γ producing CD8+ or CD4+ T cells, in cured VL or CL subjects [75, 77, 78, 80, 82]. As far as we know, no study has described the detection of both cell populations in response to stimulation by both HLA-I and -II restricted peptides in humans. However, as we have described here, Agallou and colleagues designed multi-epitope peptides containing both MHC-I and -II restricted epitopes that were able to induce both IFN-γ-producing CD4+ and CD8+ T cells in immunized mice [40]. More interestingly, it was recently reported that mice vaccination with a chimera vaccine composed of F1 and F3 NH36 domains, holding respectively, epitopes involved in both CD8+ and CD4+ T cell mediated protective responses, was more efficient than vaccination with either of the domains injected separately [67, 89].

GrB, is a proapoptotic protease that has been associated with both tissue damage and good prognosis in CL and VL patients [28–32, 95, 96]. It was recently reported that GrB constitutes a good correlate for protection against severe forms of CL [33].

Although we did not detect GrB in response to P21 peptide pool by CBA, we showed a significant increase in the percentage of CD4+GrB+ T cells after stimulation with P8, P9, P16 and P21 peptide pools, in healed CL subjects. A high frequency of CD4+ T cells producing IFN-γ and GrB was also observed in response to P21 stimulation. Interestingly, we previously showed that LmlRAB protein, from which some of our peptides are derived, was able to induce significant GrB levels in healed CL individuals [48]. Significant levels of GrB in response to HLA-A*0201-restricted T cell epitopes from *L. major* excreted/secreted or to *Leishmania*-Activated C-Kinase (LACK) proteins and a high frequency of CD4+ T cells producing GrB in response to *L. major* excreted/secreted proteins, were reported in healed CL individuals [76, 87, 97]. The simultaneous expression of GrB and IFN-γ by CD8+ T cells was also described in cured patients [96].

The importance of multifunctional T cells that produce IFN-γ, TNF-α, and IL-2 in *Leishmania* infection control has been reported [20, 21, 23, 96]. Other authors suggested that multi-cytokine production may not be a requirement for optimal function of T effector cells that mediate concomitant immunity, in the context of a persisting primary infection [98]. In this study, the multiparameter cytometry analysis revealed that peptide pools P13, P14 and P21 were able to increase significantly the frequencies of bifunctional CD4+ IFN-γ+TNF-α+ and CD4+ IL-2+TNF-α+ T cells, confirming their ability to induce a specific Th1 response in healed CL subjects. However, no increases were observed in the frequencies of multifunctional CD4+ T cells producing simultaneously IFN-γ, TNF-α and IL-2. The induction of high proportions of both CD4+ and CD8+ IFN-γ+TNF-α+ by predicted T cell epitopes from NH36 or phosphoenolpyruvate carboxykinase (PEPCK) *Leishmania* proteins was described, respectively, in infected and vaccinated mice [67, 89, 99]. However, as far as we know, no study has described the detection of multifunctional T cells in response to stimulation by HLA restricted peptides in humans.
Finally, we showed that healed CL individuals exhibited a significant increase in the percentages of CD4+ and CD8+ TCM (CD45RO+CCR7+) cells when stimulated with P8, P13, P14 and P21 peptide pools. The period of *in vitro* lymphocyte culture used here (10 days) may explain the detection of central memory cells, rather than the more short-lived effector memory cells measured in *ex vivo* assays [100]. Moreover, the same peptide pools (except P13) also induced significant levels of IFN-γ, when analyzed by the cultured ELISPOT assay, which has been described to detect central memory T cells [101, 102]. It was demonstrated in a murine model of leishmaniasis that unlike TEM, TCM are maintained in the absence of parasites and mediate long-term resistance, suggesting that these cells should be the targets for nonlive vaccines against infectious diseases requiring cell-mediated immunity [103]. TCM cells were also identified in CL patients that have healed, suggesting that these cells are generated during human leishmaniasis [35, 104]. However, it is important to emphasize that some observations in murine models of CL, based on the fact that the long-term persistence of parasites in the host is able to maintain resistance to reinfection (concomitant immunity), suggest that although TCM cells are essential components of protective immunity, they are probably not sufficient alone to induce optimal immunity required for protection against a sand fly-transmitted infection [98, 105, 106]. It has been shown that rapidly recruited, pre-existing CD4+Ly6C+T effector cells that are short-lived in the absence of infection, mediate concomitant immunity at the site of secondary infected sand fly challenge, in mice with a chronic primary infection [98, 107]. The authors suggest that the ability of a vaccine to induce an optimal protective immunity to *Leishmania* infection, may depend on the presence of both CD4+ TCM and T effector cells that should therefore be considered in vaccine development.

In this investigation, we have identified highly promiscuous HLA-I and -II restricted epitope combinations, derived from H2B, PSA and LmlRAB proteins, already involved in protection against *Leishmania* infection. These epitope combinations were able to induce IFN-γ-producing CD4+ and CD8+ T cells, GrB-producing CD4+ T cells, bifunctional CD4+ IFN-γ+TNF-α+ T cells, and CD4+ and CD8+ TCM cells, in individuals with healed CL. It is interesting to note that, among shorter peptide combinations, the P13 containing only three multi-epitope peptides composed of HLA-I and -II restricted epitopes from H2B and PSA proteins, was as efficient as P21 containing eleven multi-epitope peptides, in inducing CD4+ and CD8+ T cell responses. The data presented in this study represents a step forward in the identification of both CD4+ and CD8+ specific T cell epitopes that could be exploited for the design of a polytope vaccine against leishmaniasis.

**Supporting information**

S1 Table. HLA-typing of cured CL subjects and IFN-γ responses.
(DOCX)

S1 Fig. Gating strategy used to assess CD4+ T-cells producing IFN-γ or Granzyme B.
(PDF)

S2 Fig. Percentages of CD4+ T Cells producing IFN-γ and GrB to P21 peptide pool.
(TIF)

S3 Fig. Gating strategy used to assess CD8+ T-cells producing IFN-γ.
(PDF)

S4 Fig. Gating strategy used to assess multifunctional CD4+ T cells.
(PDF)
S5 Fig. Gating strategy used to assess CD4+ and CD8+ memory T cell.
(PDF)

**Acknowledgments**

We would like to thank blood donors for the generous donation of their cells. We also thank J. Pissarra for DNA extraction experiments. We are grateful to N. Garnaoui, A. (Amine) Garnaoui and K. Garnaoui for manuscript reading.

**Author Contributions**

**Conceptualization:** Rachel Bras-Gonçalves, Amel Meddeb-Garnaoui.

**Data curation:** Sarra Hamrouni, Rachel Bras-Gonçalves, Yasmine Messaoudi, Amel Meddeb-Garnaoui.

**Formal analysis:** Sarra Hamrouni, Rachel Bras-Gonçalves, Yasmine Messaoudi, Amel Meddeb-Garnaoui.

**Funding acquisition:** Amel Meddeb-Garnaoui.

**Investigation:** Sarra Hamrouni, Rachel Bras-Gonçalves, Rym Chamakh-Ayari, Elodie Petitdier, Yasmine Messaoudi, Julie Pagniez, Amel Meddeb-Garnaoui.

**Methodology:** Sarra Hamrouni, Rachel Bras-Gonçalves, Amel Meddeb-Garnaoui.

**Project administration:** Amel Meddeb-Garnaoui.

**Resources:** Abdelhamid Kidar, Karim Aoun.

**Supervision:** Amel Meddeb-Garnaoui.

**Validation:** Sarra Hamrouni, Rachel Bras-Gonçalves, Jean-Loup Lemesre, Amel Meddeb-Garnaoui.

**Visualization:** Sarra Hamrouni, Amel Meddeb-Garnaoui.

**Writing – original draft:** Sarra Hamrouni, Amel Meddeb-Garnaoui.

**Writing – review & editing:** Amel Meddeb-Garnaoui.

**References**

1. Burza S, Croft SL, Boelaert M. Leishmaniasis. Lancet. 2018; 392(10151): 951–70. https://doi.org/10.1016/S0140-6736(18)31204-2 PMID: 30126638

2. Courtenay O, Peters NC, Rogers ME, Bern C. Combining epidemiology with basic biology of sand flies, parasites, and hosts to inform leishmaniasis transmission dynamics and control. PLoS Pathog. 2017; 13(10): e1006571. https://doi.org/10.1371/journal.ppat.1006571 PMID: 29049371

3. Karimkhani C, Wanga V, Coffeng LE, Naghavi P, Dellavalle RP, Naghavi M. Global burden of cutaneous leishmaniasis: a cross-sectional analysis from the Global Burden of Disease Study 2013. Lancet Infect Dis. 2016; 16(5): 584–91. https://doi.org/10.1016/S1473-3099(16)00003-7 PMID: 26879176

4. Salah AB, Kamarianakis Y, Chlif S, Alaya NB, Prastacos P. Zoonotic cutaneous leishmaniasis in central Tunisia: spatio temporal dynamics. Int J Epidemiol. 2007; 36(5): 991–1000. https://doi.org/10.1093/ije/dym125 PMID: 17591639

5. Bettaieb J, Toumi A, Chlif S, Chelghaf B, Boukthir A, Gharbi A, et al. Prevalence and determinants of Leishmania major infection in emerging and old foci in Tunisia. Parasit Vectors. 2014; 7: 386. https://doi.org/10.1186/1756-3305-7-386 PMID: 25142220

6. Ponte-Sucre A, Gamarro F, Dujardin JC, Barrett MP, Lopez-Velez R, Garcia-Hernandez R, et al. Drug resistance and treatment failure in leishmaniasis: A 21st century challenge. PLoS Negl Trop Dis. 2017; 11(12): e0006052. https://doi.org/10.1371/journal.pntd.0006052 PMID: 29240765
7. Scott P, Natovitz P, Coffman RL, Pearce E, Sher A. Immunoregulation of cutaneous leishmaniasis. T cell lines that transfer protective immunity or exacerbation belong to different T helper subsets and respond to distinct parasite antigens. J Exp Med. 1988; 168(5): 1675-84. https://doi.org/10.1084/jem.168.5.1675 PMID: 2903212

8. Sacks D, Noben-Trauth N. The immunology of susceptibility and resistance to Leishmania major in mice. Nat Rev Immunol. 2002; 2(11): 845-58. https://doi.org/10.1038/nri933 PMID: 12415308

9. Scott P, Novais FO. Cutaneous leishmaniasis: immune responses in protection and pathogenesis. Nat Rev Immunol. 2016; 16(9): 581-92. https://doi.org/10.1038/nri.2016.72 PMID: 27424773

10. Ajdary S, Alimohammadian MH, Eslami MB, Kemp K, Kharazmi A. Comparison of the immune profile of nonhealing cutaneous Leishmaniasis patients with those with active lesions and those who have recovered from infection. Infect Immun. 2000; 68(4): 1760-4. https://doi.org/10.1128/iai.68.4.1760-1764.2000 PMID: 10722561

11. Kammoun-Rebai W, Naouar I, Libri V, Albert M, Louzir H, Meddeb-Garnaoui A, et al. Protein biomarkers discriminate Leishmania major-infected and non-infected individuals in areas endemic for cutaneous leishmaniasis. BMC Infect Dis. 2016; 16: 138. https://doi.org/10.1186/s12879-016-1458-6 PMID: 27009263

12. Sassi A, Louzir H, Ben Salah A, Mokni M, Ben Osman A, Dellagi K. Leishmanin skin test lymphoproliferative responses and cytokine production after symptomatic or asymptomatic Leishmania major infection in Tunisia. Clin Exp Immunol. 1999; 116(1): 127-32. https://doi.org/10.1046/j.1365-2249.1999.00844.x PMID: 10209516

13. Ben Salah A, Louzir H, Chlif S, Mokni M, Zaatour A, Raouene M, et al. The predictive validity of naturally acquired delayed-type hypersensitivity to leishmanin in resistance to Leishmania major-associated cutaneous leishmaniasis. J Infect Dis. 2005; 192(11): 1981-7. https://doi.org/10.1086/498042 PMID: 16267771

14. Kemp K, Theander TG, Hviid L, Garfar A, Kharazmi A, Kemp M. Interferon-gamma- and tumour necrosis factor-alpha-producing cells in humans who are immune to cutaneous leishmaniasis. Scand J Immunol. 1999; 49(6): 655-9. https://doi.org/10.1046/j.1365-3083.1999.00554.x PMID: 10354378

15. Louzir H, Melby PC, Ben Salah A, Marrakchi H, Aoun K, Ben Ismail R, et al. Immunologic determinants of disease evolution in localized cutaneous leishmaniasis due to Leishmania major. J Infect Dis. 1998; 177(6): 1687-95. https://doi.org/10.1086/515297 PMID: 9607850

16. Bourreau E, Prevot G, Gardon J, Pradinaud R, Launois P. High intralesional interleukin-10 messenger RNA expression in localized cutaneous leishmaniasis is associated with unresponsiveness to treatment. J Infect Dis. 2001; 184(12): 1628-30. https://doi.org/10.1086/324665 PMID: 11740743

17. Bourreau E, Prevot G, Gardon J, Pradinaud R, Hasagewa H, Milon G, et al. LACK-specific CD4(+) T cells that induce gamma interferon production in patients with localized cutaneous leishmaniasis during an early stage of infection. Infect Immun. 2002; 70(6): 3122-9. https://doi.org/10.1128/IAI.70.6.3122-3129.2002 PMID: 12011006

18. Anderson CF, Oukka M, Kuchroo VJ, Sacks D. CD4(+)CD25(-)Foxp3(-) Th1 cells are the source of IL-10-mediated immune suppression in chronic cutaneous leishmaniasis. J Exp Med. 2007; 204(2): 285-97. https://doi.org/10.1084/jem.20061886 PMID: 17283207

19. Boussoffara T, Boubaker MS, Ben Ahmed M, Mokni M, Guizani I, Ben Salah A, et al. Histological and immunological differences between zoonotic cutaneous leishmaniasis due to Leishmania major and sporadic cutaneous leishmaniasis due to Leishmania infantum. Parasite. 2019; 26: 9. https://doi.org/10.1051/parasite/2019007 PMID: 30810524

20. Darrah PA, Patel DT, De Luca PM, Lindsay RW, Davey DF, Flynn BJ, et al. Multifunctional TH1 cells define a correlate of vaccine-mediated protection against Leishmania major. Nat Med. 2007; 13(7): 843-50. https://doi.org/10.1038/nm1592 PMID: 17558415

21. Macedo AB, Sanchez-Arcila JC, Schubach AO, Mendonca SC, Marins-Dos-Santos A, de Fatima Madeira M, et al. Multifunctional CD4(+) T cells in patients with American cutaneous leishmaniasis. Clin Exp Immunol. 2012; 167(3): 505-13. https://doi.org/10.1111/j.1365-2249.2011.04536.x PMID: 22288594

22. Nico D, Gomes DC, Palatnik-de-Sousa I, Morrot A, Palatnik M, Palatnik-de-Sousa CB. Leishmania donovani Nucleoside Hydrolase Terminal Domains in Cross-Protective Immunotherapy Against Leishmania amazonensis Murine Infection. Front Immunol. 2014; 5: 273. https://doi.org/10.3389/fimmu.2014.00273 PMID: 24966857

23. Barbosa Santos ML, Nico D, de Oliveira FA, Barreto AS, Palatnik-de-Sousa I, Carrillo E, et al. Leishmania donovani Nucleoside Hydrolase (NH36) Domains Induce T-Cell Cytokine Responses in Human Visceral Leishmaniasis. Front Immunol. 2017; 8: 227. https://doi.org/10.3389/fimmu.2017.00227 PMID: 28321221
24. da Silva Santos C, Brodskyn CI. The Role of CD4 and CD8 T Cells in Human Cutaneous Leishmania-sis. Front Public Health. 2014; 2: 165. https://doi.org/10.3389/fpubh.2014.00165 PMID: 25325049

25. Da-Cruz AM, Bittar R, Mattos M, Oliveira-Neto MP, Nogueira R, Pinho-Ribeiro V, et al. T-cell-mediated immune responses in patients with cutaneous or mucosal leishmaniasis: long-term evaluation after therapy. Clin Diagn Lab Immunol. 2002; 9(2): 251-6. https://doi.org/10.1128/CDLI.9.2.251-256.2002 PMID: 11874860

26. Nateghi Rostami M, Keshavarz H, Edalat R, Sarrafnejad A, Shahrestani T, Mahboudi F, et al. CD8+ T cells as a source of IFN-gamma production in human cutaneous leishmaniasis. PLoS Negl Trop Dis. 2010; 4(10): e845. https://doi.org/10.1371/journal.pntd.0000845 PMID: 20967288

27. Santos Cda S, Boaventura V, Ribeiro Cardoso C, Tavares N, Lordelo MJ, Noronha A, et al. CD8(+) granzyme B(+)-mediated tissue injury vs. CD4(+)IFNgamma(+)mediated parasite killing in human cutaneous leishmaniasis. J Invest Dermatol. 2013; 133(6): 1533-40. https://doi.org/10.1038/jid.2013.4 PMID: 23321919

28. da Silva Santos C, Attarha S, Saini RK, Boaventura V, Costa J, Khouri R, et al. Proteome profiling of human cutaneous leishmaniasis lesion. J Invest Dermatol. 2015; 135(2): 400-10. https://doi.org/10.1038/jid.2014.396 PMID: 25207817

29. Lago TS, Silva JA, Lago EL, Carvalho EM, Zanette DL, Castellucci LC. The miRNA 361-3p, a Regulator of GZMB and TNF Is Associated With Therapeutic Failure and Longer Time Healing of Cutaneous Leishmaniasis Caused by L. (viannia) braziliensis. Front Immunol. 2018; 9: 2621. https://doi.org/10.3389/fimmu.2018.02621 PMID: 30487794

30. Cardoso TM, Machado A, Costa DL, Carvalho LP, Queiroz A, Machado P, et al. Protective and pathological functions of CD8+ T cells in Leishmania braziliensis infection. Infect Immun. 2015; 83(3): 898-906. https://doi.org/10.1128/IAI.02404-14 PMID: 25534940

31. 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. https://doi.org/10.1086/382031 PMID: 15031796

32. Kaushal H, Bras-Goncalves R, Negi NS, Lemesre JL, Papierok G, Salotra P. Role of CD8(+) T cells in protection against Leishmania donovani infection in healed Visceral Leishmaniasis individuals. BMC Infect Dis. 2014; 14: 653. https://doi.org/10.1186/s12879-014-0653-6 PMID: 25471494

33. Bousoffara T, Chelif S, Ben Ahmed M, Mokni M, Ben Salah A, Dellagi K, et al. Immunity Against Leishmania major Infection: Parasite-Specific Granzyme B Induction as a Correlate of Protection. Front Cell Infect Microbiol. 2018; 8: 397. https://doi.org/10.3389/fcimb.2018.00397 PMID: 30483482

34. Carvalho AM, Magalhaes A, Carvalho LP, Bacellar O, Scott P, Carvalho EM. Immunologic response and memory T cells in subjects cured of tegumentary leishmaniasis. BMC Infect Dis. 2013; 13: 529. https://doi.org/10.1186/1471-2334-13-529 PMID: 24206576

35. Keshavarz Valian H, Nateghi Rostami M, Tasbihi M, Miramin Mohammadi A, Eskandari SE, Sarrafnejad A, et al. CCR7+ central and CCR7- effector memory CD4+ T cells in human cutaneous leishmaniasis. J Clin Immunol. 2013; 33(1): 220-34. https://doi.org/10.1007/s10875-012-9788-7 PMID: 22990666

36. Khamesipour A, Nateghi Rostami M, Tasbihi M, Miramin Mohammadi A, Shahrestani T, Sarrafnejad A, et al. Phenotyping of circulating CD8(+) T cell subsets in human cutaneous leishmaniasis. Microbes Infect. 2012; 14(9): 702-11. https://doi.org/10.1016/j.micinf.2012.02.006 PMID: 22421108

37. Duthie MS, Raman VS, Piazza FM, Reed SG. The development and clinical evaluation of second-generation leishmaniasis vaccines. Vaccine. 2012; 30(2): 134-41. https://doi.org/10.1016/j.vaccine.2011.11.005 PMID: 22085553

38. Iborra S, Solana JC, Requena JM, Soto M. Vaccine candidates against leishmania under current research. Expert Rev Vaccines. 2018; 17(4): 323-34. https://doi.org/10.1080/14760584.2018.1459191 PMID: 29589966

39. De Groot AS, McMurry J, Marcon L, Franco J, Rivera D, Kutzler M, et al. Developing an epitope-driven tuberculosis (TB) vaccine. Vaccine. 2005; 23(17-18): 2121-31. https://doi.org/10.1016/j.vaccine.2005.01.059 PMID: 15755582

40. Agallou M, Athanasiou E, Koutsoni O, Dotsika E, Karagouni E. Experimental Validation of Multi-Epitope Peptides Including Promising MHC Class I- and II-Restricted Epitopes of Four Known Leishmania infantum Proteins. Front Immunol. 2014; 5: 268. https://doi.org/10.3389/fimmu.2014.00268 PMID: 24959167

41. Seyed N, Taheri T, Rafati S. Post-Genomics and Vaccine Improvement for Leishmania. Front Microbiol. 2016; 7: 467. https://doi.org/10.3389/fmicb.2016.00467 PMID: 27092123

42. Barve M, Bender J, Senzer N, Cunningham C, Greco FA, McCune D, et al. Induction of immune responses and clinical efficacy in a phase II trial of IDM-2101, a 10-epitope cytotoxic T-lymphocyte
vaccine, in metastatic non-small-cell lung cancer. J Clin Oncol. 2008; 26(27): 4418-25. https://doi.org/
10.1200/JCO.2008.16.6462 PMID: 18802154

43. Nardin E. The past decade in malaria synthetic peptide vaccine clinical trials. Hum Vaccin. 2010; 6(1): 27-38. https://doi.org/10.4161/hv.6.1.9601 PMID: 20173408

44. Rosendahl Huber S, van Beek J, de Jonge J, Luytjes W, van Baarle D. T cell responses to viral infections—opportunities for Peptide vaccination. Front Immunol. 2014; 5: 171. https://doi.org/10.3389/fimmu.2014.00171 PMID: 24795718

45. Serna C, Lara JA, Rodrigues SP, Marques AF, Almeida IC, Maldonado RA. A synthetic peptide from Trypanosoma cruzi mucin-like associated surface protein as candidate for a vaccine against Chagas disease. Vaccine. 2014; 32(28): 3525-32. https://doi.org/10.1016/j.vaccine.2014.04.026 PMID: 24793944

46. Li W, Joshi MD, Singhania S, Ramsey KH, Murthy AK. Peptide Vaccine: Progress and Challenges. Vaccines (Basel). 2014; 2(3): 515-36.

47. Chamakh-Ayari R, Bras-Goncalves R, Bahi-Jaber N, Petitdidier E, Markikou-Ouni W, Aoun K, et al. In vitro evaluation of a soluble Leishmania promastigote surface antigen as a potential vaccine candidate against human leishmaniasis. PLoS One. 2014; 9(5): e92708. https://doi.org/10.1371/journal.pone.0092708 PMID: 24786587

48. Chamakh-Ayari R, Chenik M, Chakroun AS, Bahi-Jaber N, Aoun K, Meddeb-Garnaoui A. Leishmania major large RAB GTPase is highly immunogenic in individuals immune to cutaneous and visceral leishmaniasis. Parasit Vectors. 2017; 10(1): 185. https://doi.org/10.1186/s13071-017-2127-3 PMID: 28416006

49. Meddeb-Garnaoui A, Toumi A, Ghelis H, Mahjoub M, Louzir H, Chenik M. Cellular and humoral responses induced by Leishmania histone H2B and its divergent and conserved parts in cutaneous and visceral leishmaniasis patients, respectively. Vaccine. 2010; 28(7): 1881-6. https://doi.org/10.1016/j.vaccine.2009.11.075 PMID: 20005858

50. Petitdidier E, Pagniez J, Papierok G, Vincendeau P, Lemesre JL, Bras-Goncalves R. Recombinant Forms of Leishmania amazonensis Excreted/Secreted Promastigote Surface Antigen (PSA) Induce Protective Immune Responses in Dogs. PLoS Negl Trop Dis. 2016; 10(5): e0004614. https://doi.org/10.1371/journal.pntd.0004614 PMID: 27223609

51. Chenik M, Louzir H, Ksontini H, Dilou A, Abdmouleh I, Dellagi K. Vaccination with the divergent portion of the protein histone H2B of Leishmania protects susceptible BALB/c mice against a virulent challenge with Leishmania major. Vaccine. 2006; 24(14): 2521-9. https://doi.org/10.1016/j.vaccine.2005.12.027 PMID: 16417957

52. Handman E, Symons FM, Baldwin TM, Curtis JM, Scheerlinck JP. Protective vaccination with promastigote surface antigen 2 from Leishmania major is mediated by a TH1 type of immune response. Infect Immun. 1995; 63(11): 4261-7. PMID: 7591056

53. Taylor WR. The classification of amino acid conservation. J Theor Biol. 1986; 119(2): 205-18. https://doi.org/10.1016/s0022-5193(86)80075-3 PMID: 3461222

54. Sidney J, Peters B, Frahm N, Brander C, Sette A. HLA class I supertypes: a revised and updated classification. BMC Immunol. 2008; 9: 1. https://doi.org/10.1186/1471-2172-9-1 PMID: 18211710

55. Lund O, Nielsen M, Kesmir C, Petersen AG, Lundegaard C, Worning P, et al. Definition of supertypes for HLA molecules using clustering of specificity matrices. Immunogenetics. 2004; 55(12): 797-810. https://doi.org/10.1007/s00251-004-0647-4 PMID: 14963618

56. Sette A, Newman M, Livingston B, McKinney D, Sidney J, Ishioka G, et al. Optimizing vaccine design for cellular processing, MHC binding and TCR recognition. Tissue Antigens. 2002; 59(6): 443-51. https://doi.org/10.1034/j.1399-0039.2002.590601.x PMID: 12445314

57. Cardinaud S, Bouziat R, Rohrlich PS, Tourdot S, Weiss L, Langlade-Demoyen P, et al. Design of a HIV-1-derived HLA-B07.02-restricted polypepitope construct. AIDS. 2009; 23(15): 1945-54. https://doi.org/10.1097/QAD.0b013e32832fae88 PMID: 19644347

58. Levy A, Pitcovski J, Frankenburg S, Elias O, Altuvia Y, Margalit H, et al. A melanoma multiepitope polypeptide induces specific CD8+ T-cell response. Cell Immunol. 2007; 250(1-2): 24-30. https://doi.org/10.1016/j.cellimm.2008.01.001 PMID: 18275944

59. Greenbaum J, Sidney J, Chung J, Brander C, Peters B, Sette A. Functional classification of class II human leukocyte antigen (HLA) molecules reveals seven different supertypes and a surprising degree of repertoire sharing across supertypes. Immunogenetics. 2011; 63(6): 325-35. https://doi.org/10.1007/s00251-011-0513-0 PMID: 21305276

60. Loing E, Andrieu M, Thiam K, Schorner D, Wiesmuller KH, Hosmalin A, et al. Extension of HLA-A*0201-restricted minimal epitope by N epsilon-palmitoyl-lysine increases the life span of functional presentation to cytotoxic T cells. J Immunol. 2000; 164(2): 900-7. https://doi.org/10.4049/jimmunol.164.2.900 PMID: 10623838
61. Baier W, Masihi N, Huber M, Hoffmann P, Bessler WG. Lipopeptides as immunoadjuvants and immunostimulants in mucosal immunization. Immunobiology. 2000; 201(3-4): 391-405. https://doi.org/10.1016/s0171-2985(00)80093-5 PMID: 10776795

62. Moyle PM, Toth I. Self-adjuvanting lipopeptide vaccines. Curr Med Chem. 2008; 15(5): 506-16. https://doi.org/10.2174/092986708783503249 PMID: 18289006

63. Launay O, Durier C, Desaint C, Silbermann B, Jackson A, Pialoux G, et al. Cellular immune responses induced with dose-sparing intradermal administration of HIV vaccine to HIV-uninfected volunteers in the ANRS VAC16 trial. PLoS One. 2007; 2(8): e725. https://doi.org/10.1371/journal.pone.0000725 PMID: 17712402

64. Bras-Goncalves R, Petitdidier E, Pagniez J, Veyrier R, Cibrelus P, Cavaleyra M, et al. Identification and characterization of new Leishmania promastigote surface antigens, LaPSA-38S and LiPSA-50S, as major immunodominant excreted/secreted components of L. amazonensis and L. infantum. Infect Genet Evol. 2014; 24: 1-14. https://doi.org/10.1016/j.meegid.2014.02.017 PMID: 24614507

65. Purcell AW, McCluskey J, Rossjohn J. More than one reason to rethink the use of peptides in vaccine design. Nat Rev Drug Discov. 2007; 6(5): 404-14. https://doi.org/10.1038/nrd2224 PMID: 17473845

66. Skwarczynski M, Toth I. Peptide-based synthetic vaccines. Chem Sci. 2016; 7(2): 842-54. https://doi.org/10.1039/c5sc03892h PMID: 28791117

67. Alves-Silva MV, Nico D, Morrot A, Palatnik M, Palatnik-de-Sousa CB. A Chimera Containing CD4+ and CD8+ T-Cell Epitopes of the Leishmania donovani Nucleoside Hydrolase (NH36) Optimizes Cross-Protection against Leishmania amazonensis Infection. Front Immunol. 2017; 8: 100. https://doi.org/10.3389/fimmu.2017.00100 PMID: 28280494

68. De Brito RCF, Cardoso JMO, Reis LES, Vieira JF, Mathias FAS, Roatt BM, et al. Peptide Vaccines for Leishmaniasis. Front Immunol. 2018; 9: 1043. https://doi.org/10.3389/fimmu.2018.01043 PMID: 29868006

69. Iborra S, Soto M, Carrion J, Alonso C, Requena JM. Vaccination with a plasmid DNA cocktail encoding the nucleosomal histones of Leishmania confers protection against murine cutaneous leishmaniosis. Vaccine. 2004; 22(29-30): 3865-76. https://doi.org/10.1016/j.vaccine.2004.04.015 PMID: 15364433

70. Chenik M, Chaabouni N, Ben Achour-Chenik Y, Ouakad M, Lakhal-Naouar I, Louzir H, et al. Identification of a new developmentally regulated Leishmania major large RAB GTPase. Biochem Biophys Res Commun. 2006; 341(2): 541-8. https://doi.org/10.1016/j.bbrc.2006.01.005 PMID: 16430865

71. Peters NC, Kimblin N, Secundino N, Kamhawi S, Lawyer P, Sacks DL. Vector transmission of leishmania abrogates vaccine-induced protective immunity. PLoS Pathog. 2009; 5(6): e1000484. https://doi.org/10.1371/journal.ppat.1000484 PMID: 19543375

72. Peters NC, Sacks DL. The impact of vector-mediated neutrophil recruitment on cutaneous leishmaniasis. Cell Microbiol. 2009; 11(9): 1290-6. https://doi.org/10.1111/j.1462-5822.2009.01348.x PMID: 19545276

73. Peters NC, Bertholet S, Lawyer PG, Charmoy M, Romano A, Ribeiro-Gomes FL, et al. Evaluation of recombinant Leishmania polyprotein plus glucopyranosyl lipid A stable emulsion vaccines against sand fly-transmitted Leishmania major in C57BL/6 mice. J Immunol. 2012; 189(10): 4832-41. https://doi.org/10.4049/jimmunol.1201676 PMID: 23045616

74. Saffari B, Mohabatkar H. Computational analysis of cysteine proteases (Clan CA, Family Cl) of Leishmania major to find potential epitopic regions. Genomics Proteomics Bioinformatics. 2009; 7(3): 87-95. https://doi.org/10.1016/S1672-0229(08)60037-6 PMID: 19944381

75. 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 T cell response. PLoS Negl Trop Dis. 2011; 5(9): e1295. https://doi.org/10.1371/journal.pntd.0001295 PMID: 21909442

76. Naouar I, Boussoffara T, Chenik M, Gritli S, Ben Ahmed M, Belhadj Hmida N, et al. Prediction of T Cell Epitopes from Leishmania major Potentially Excreted/Secreted Proteins Inducing Granzyme B Production. PLoS One. 2016; 11(1): e0147076. https://doi.org/10.1371/journal.pone.0147076 PMID: 26771180

77. Dikhit MR, Amit A, Singh AK, Kumar A, Mansuri R, Sinha S, et al. Vaccine potential of HLA-A2 epitopes from Leishmania Cysteine Protease Type III (CPC). Parasite Immunol. 2017; 39(9).

78. Vijayamahantesh, Amit A, Dikhit MR, Singh AK, Venkateshwaran T, Das VNR, et al. Immuno-informatics based approaches to identify CD8+ T cell epitopes within the Leishmania donovani 3-ectonucleotidase in cured visceral leishmaniasis subjects. Microbes Infect. 2017; 19(6): 358-69. https://doi.org/10.1016/j.micinf.2017.03.002 PMID: 28373107

79. Amit A, Dikhit MR, Mahantesh V, Chaudhary R, Singh AK, Singh A, et al. Immunomodulation mediated through Leishmania donovani protein disulfide isomerase by eliciting CD8+ T-cell in cured visceral
leishmaniasis subjects and identification of its possible HLA class-1 restricted T-cell epitopes. J Biomol Struct Dyn. 2017; 35(1): 128–40. https://doi.org/10.1080/07391102.2015.1134349 PMID: 26727289

80. Dikhit MR, Vijayamahantesh, Kumar A, Amit A, Dehury B, Nathsharma YP, et al. Mining the Proteome of Leishmania donovani for the Development of Novel MHC Class I Restricted Epitope for the Control of Visceral Leishmaniasis. J Cell Biochem. 2018; 119(1): 378–91. https://doi.org/10.1002/jcb.26190 PMID: 28585770

81. Elfaki ME, Khalil EA, De Groot AS, Musa AM, Gutierrez A, Younis BM, et al. Immunogenicity and immune modulatory effects of in silico predicted L. donovani candidate peptide vaccines. Hum Vaccin Immunother. 2012; 8(12): 1769–74. https://doi.org/10.4161/hv.21881 PMID: 22922767

82. Dikhit MR, Kumar A, Das S, Dehury B, Rout AK, Jamal F, et al. Identification of Potential MHC Class-II-Restricted Epitopes Derived from Leishmania donovani Antigens by Reverse Vaccinology and Evaluation of Their CD4+ T-Cell Responsiveness against Visceral Leishmaniasis. Front Immunol. 2017; 8: 1763. https://doi.org/10.3389/fimmu.2017.01763 PMID: 29312304

83. Joshi S, Yadav NK, Rawat K, Kumar V, Ali R, Sahasrabuddhe AA, et al. Immunogenicity and Protective Efficacy of T-Cell Epitopes Derived From Potential Th1 Stimulatory Proteins of Leishmania (Leishmania) donovani. Front Immunol. 2019; 10: 288. https://doi.org/10.3389/fimmu.2019.00288 PMID: 30873164

84. Silva E, Ferreira LF, Hernandes MZ, de Brito ME, de Oliveira BC, da Silva AA, et al. Combination of In Silico Methods in the Search for Potential CD4(+) and CD8(+) T Cell Epitopes in the Proteome of Leishmania braziliensis. Front Immunol. 2016; 7: 327. https://doi.org/10.3389/fimmu.2016.00327 PMID: 27621732

85. Dias DS, Ribeiro PAF, Martins VT, Lage DP, Costa LE, Chavez-Fumagalli MA, et al. Vaccination with a CD4(+) and CD8(+) T-cell epitopes-based recombinant chimeric protein derived from Leishmania infantum proteins confers protective immunity against visceral leishmaniasis. Transl Res. 2018; 200: 18–34. https://doi.org/10.1016/j.trsl.2018.05.001 PMID: 29908151

86. Kima PE, Soong L. Interferon gamma in leishmaniasis. Front Immunol. 2013; 4: 156. https://doi.org/10.3389/fimmu.2013.00156 PMID: 23801993

87. 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; 2014: 636039. https://doi.org/10.1155/2014/636039 PMID: 25104882

88. Nico D, Gomes DC, Alves-Silva MV, Freitas EO, Morrot A, Bahia D, et al. Cross-Protective Immunity to Leishmania amazonensis is Mediated by CD4+ and CD8+ Epitopes of Leishmania donovani Nucleoside Hydrolase Terminal Domains. Front Immunol. 2014; 5: 189. https://doi.org/10.3389/fimmu.2014.00189 PMID: 24822054

89. Alves-Silva MV, Nico D, de Luca PM, Palatnik de-Sousa CB. The F1F3 Recombinant Chimera of Leishmania donovani-Nucleoside Hydrolase (NH36) and Its Epitopes Induce Cross-Protection Against Leishmania (V.) braziliensis Infection in Mice. Front Immunol. 2019; 10: 724. https://doi.org/10.3389/fimmu.2019.00724 PMID: 31024556

90. Kumar P, Misra P, Thakur CP, Saurabh A, Rishi N, Mitra DK. T cell suppression in the bone marrow of visceral leishmaniasis patients: impact of parasite load. Clin Exp Immunol. 2018; 191(3): 318–27. https://doi.org/10.1111/cei.13074 PMID: 29058314

91. Martins VT, Duarte MC, Lage DP, Costa LE, Carvalho AM, Mendes TA, et al. A recombinant chimeric protein composed of human and mice-specific CD4(+) and CD8(+) T-cell epitopes protects against visceral leishmaniasis. Parasite Immunol. 2017; 39(1).

92. Castelli FA, Leleu M, Pouvelle-Moratille S, Farci S, Zarour HM, Andrieu M, et al. Differential capacity of T cell priming in naive donors of promiscuous CD4+ T cell epitopes of HCV NS3 and Core proteins. Eur J Immunol. 2007; 37(6): 1513–23. https://doi.org/10.1002/eji.200636783 PMID: 17492804

93. Hamze M, Meunier S, Karle A, Gdoura A, Goudet A, Szely N, et al. Characterization of CD4 T Cell Epitopes of Infliximab and Rituximab Identified from Healthy Donors. Front Immunol. 2017; 8: 500. https://doi.org/10.3389/fimmu.2017.00500 PMID: 28529511

94. Bottrel RL, Dutra WO, Martins FA, Gontijo B, Carvalho E, Barral-Netto M, et al. Flow cytometric determination of cellular sources and frequencies of key cytokine-producing lymphocytes directed against recombinant LACK and soluble Leishmania antigen in human cutaneous leishmaniasis. Infect Immun. 2001; 69(5): 3232–9. https://doi.org/10.1128/IAI.69.5.3232-3239.2001 PMID: 11292745

95. Kaushal H, Bras-Goncalves R, Avishek K, Kumar Deep D, Petitdidier E, Lemesre JL, et al. Evaluation of cellular immunological responses in mono- and polymorphic clinical forms of post-kala-azar dermal leishmaniasis in India. Clin Exp Immunol. 2016; 185(1): 50–60. https://doi.org/10.1111/cei.12787 PMID: 26948150

96. Egui A, Ledesma D, Perez-Anton E, Montoya A, Gomez I, Robledo SM, et al. Phenotypic and Functional Profiles of Antigen-Specific CD4(+) and CD8(+) T Cells Associated With Infection Control in
Patients With Cutaneous Leishmaniasis. Front Cell Infect Microbiol. 2018; 8: 393. https://doi.org/10.3389/fcimb.2018.00393 PMID: 30510917

97. Fernandez L, Carrillo E, Sanchez-Sampedro L, Sanchez C, Ibarra-Meneses AV, Jimenez MA, et al. Antigenicity of Leishmania-Activated C-Kinase Antigen (LACK) in Human Peripheral Blood Mononuclear Cells, and Protective Effect of Prime-Boost Vaccination With pCI-neo-LACK Plus Attenuated LACK-Expressing Vaccinia Viruses in Hamsters. Front Immunol. 2018; 9: 843. https://doi.org/10.3389/fimmu.2018.00843 PMID: 29740446

98. Peters NC, Pagan AJ, Lawyer PG, Hand TW, Henrique Roma E, Stamper LW, et al. Chronic parasitic infection maintains high frequencies of short-lived Ly6C+CD4+ effector T cells that are required for protection against re-infection. PLoS Pathog. 2014; 10(12): e1004538. https://doi.org/10.1371/journal.ppat.1004538 PMID: 25473946

99. Mou Z, Li J, Boussoffara T, Kishi H, Hamana H, Ezzati P, et al. Identification of broadly conserved cross-species protective Leishmania antigen and its responding CD4+ T cells. Sci Transl Med. 2015; 7(310): 310ra167. https://doi.org/10.1126/scitranslmed.aac5477 PMID: 26491077

100. Lanzavecchia A, Sallusto F. Understanding the generation and function of memory T cell subsets. Curr Opin Immunol. 2005; 17(3): 326-32. https://doi.org/10.1016/j.coi.2005.04.010 PMID: 15886125

101. Reece WH, Pinder M, Gothard PK, Milligan P, Bojang K, Doherty T, et al. A CD4(+) T-cell immune response to a conserved epitope in the circumsporozoite protein correlates with protection from natural Plasmodium falciparum infection and disease. Nat Med. 2004; 10(4): 406-10. https://doi.org/10.1038/nm1009 PMID: 15034567

102. Calarota SA, Baldanti F. Enumeration and characterization of human memory T cells by enzyme-linked immunospot assays. Clin Dev Immunol. 2013; 2013: 637649. https://doi.org/10.1155/2013/637649 PMID: 24319467

103. Zaph C, Uzonna J, Beverley SM, Scott P. Central memory T cells mediate long-term immunity to Leishmania major in the absence of persistent parasites. Nat Med. 2004; 10(10): 1104-10. https://doi.org/10.1038/nm1108 PMID: 15448686

104. Glennie ND, Scott P. Memory T cells in cutaneous leishmaniasis. Cell Immunol. 2016; 309: 50-4. https://doi.org/10.1016/j.cellimm.2016.07.010 PMID: 27493096

105. Uzonna JE, Wei G, Yurkowski D, Bretscher P. Immune elimination of Leishmania major in mice: implications for immune memory, vaccination, and reactivation disease. J Immunol. 2001; 167(12): 6967-74. https://doi.org/10.4049/jimmunol.167.12.6967 PMID: 11739516

106. Belkaid Y, Piccirillo CA, Mendez S, Shevach EM, Sacks DL. CD4+CD25+ regulatory T cells control Leishmania major persistence and immunity. Nature. 2002; 420(6915): 502-7. https://doi.org/10.1038/nature01152 PMID: 12466842

107. Hohman LS, Peters NC. CD4(+) T Cell-Mediated Immunity against the Phagosomal Pathogen Leishmania: Implications for Vaccination. Trends Parasitol. 2019; 35(6): 423-35. https://doi.org/10.1016/j.pt.2019.04.002 PMID: 31080088