Rym Chamakh-Ayari

Rachel Bras-Gonc ¸alves

Narges Bahi-Jaber

Elodie Petitdidier

Wafa Markikou-Ouni

Karim Aoun

Javier Moreno

Eugenia Carrillo

Poonam Salotra

Himanshu Kaushal

Narender Singh Negi

Jorge Arevalo

Francesca Falconi-Agapito

Angela Privat

Maria Cruz

Julie Pagniez

Ge ´rard-Marie Papierok

Bel Haj Faten

Rhouma

Pilar Torres

Jean-Loup Lemesre

Mehdi Chenik

Amel Meddeb-Garnaoui

In Vitro Evaluation of a Soluble Leishmania Promastigote Surface Antigen as a Potential Vaccine Candidate against Human Leishmaniasis

PSA (Promastigote Surface Antigen) belongs to a family of membrane-bound and secreted proteins present in several Leishmania (L.) species. PSA is recognized by human Th1 cells and provides a high degree of protection in vaccinated mice. We evaluated humoral and cellular immune responses induced by a L. amazonensis PSA protein (LaPSA-38S) produced in a L. tarentolae expression system. This was done in individuals cured of cutaneous leishmaniasis due to L. major (CCLm) or L. braziliensis (CCLb) or visceral leishmaniasis due to L. donovani (CVLd) and in healthy individuals. Healthy individuals were subdivided into immune (HHR-Lm and HHR-Li: Healthy High Responders living in an endemic area for L. major or L. infantum infection) or non immune/naive individuals (HLR: Healthy Low Responders), depending on whether they produce high or low levels of IFN-c in response to Leishmania soluble antigen. Low levels of total IgG antibodies to LaPSA-38S were detected in sera from the studied groups. Interestingly, LaPSA-38S induced specific and significant levels of IFN-c, granzyme B and IL-10 in CCLm, HHR-Lm and HHR-Li groups, with HHR-Li group producing TNF-a in more. No significant cytokine response was observed in individuals immune to L. braziliensis or L. donovani infection. Phenotypic analysis showed a significant increase in CD4+ T cells producing IFN-c after LaPSA-38S stimulation, in CCLm. A high positive correlation was observed between the percentage of IFN-c-producing CD4+ T cells and the released IFN-c. We showed that the LaPSA-38S protein was able to induce a mixed Th1 and Th2/Treg cytokine response in individuals with immunity to L. major or L. infantum infection indicating that it may be exploited as a vaccine candidate. We also showed, to our knowledge for the first time, the capacity of Leishmania PSA protein to induce granzyme B production in humans with immunity to L. major and L. infantum infection.

Introduction

Leishmania (L.) are intracellular protozoa that cause a wide spectrum of human diseases, ranging from self-healing cutaneous to lethal visceral leishmaniasis. Cutaneous leishmaniasis (CL) is widely distributed in the Americas, North Africa, the Middle East and Central Asia, causing considerable morbidity [START_REF] Alvar | Leishmaniasis worldwide and global estimates of its incidence[END_REF]. In Tunisia, CL is zoonotic and is mainly due to L. major [START_REF] Salah | Zoonotic cutaneous leishmaniasis in central Tunisia: spatio temporal dynamics[END_REF]. In Peru, L. braziliensis is responsible for nearly all the zoonotic CL cases [START_REF] Lucas | Geographic distribution and clinical description of leishmaniasis cases in Peru[END_REF].

Anthroponotic Visceral Leishmaniasis (VL) due to L. donovani is mainly distributed in the Indian subcontinent with over 300 000 annual cases [START_REF] Stauch | Visceral leishmaniasis in the Indian subcontinent: modelling epidemiology and control[END_REF], whereas zoonotic VL caused by L. infantum is present mainly in Mediterranean basin, Middle east, central Asia and Brazil with up to 50 000 annual cases [START_REF] Aoun | Current epidemiological data on visceral leishmaniasis in Tunisia][END_REF][START_REF] Den Boer | Leishmaniasis impact and treatment access[END_REF][START_REF] Marty | Mediterranean visceral leishmaniasis][END_REF]. Treatment of leishmaniasis mainly relies on chemotherapy and the control of the disease is challenged by serious side effects of existing drugs and the widespread emergence of drug resistant strains. Several lines of evidence are in favor of the feasibility of a vaccine in humans.

Healing is generally associated with the development of a life-long immunity to re-infection. Moreover, a large majority of immunocompetent exposed individuals develop asymptomatic or subclinical infections rather than a severe form of leishmaniasis (symptomatic) and acquires a robust and durable immunity to reinfection. Resistance to infection is based on a Th1 dominant response with interferon (IFN)-c production resulting in macrophage activation and parasite killing [START_REF] Sacks | The immunology of susceptibility and resistance to Leishmania major in mice[END_REF][START_REF] Mansueto | Immunopathology of leishmaniasis: an update[END_REF][START_REF] Nylen | Immunological perspectives of leishmaniasis[END_REF]. In healed individuals, it has been shown that recovery and resistance to re-infection also correlate with the development of antigen-specific Th1 cell responses and IFN-c production [START_REF] Carvalho | Characterization of the immune response in subjects with self-healing cutaneous leishmaniasis[END_REF][START_REF] Kharazmi | T-cell response in human leishmaniasis[END_REF][START_REF] Sassi | Leishmanin skin test lymphoproliferative responses and cytokine production after symptomatic or asymptomatic Leishmania major infection in Tunisia[END_REF][START_REF] Ajdary | Comparison of the immune profile of nonhealing cutaneous Leishmaniasis patients with those with active lesions and those who have recovered from infection[END_REF]. Based on these data, one vaccine strategy developed against Leishmania infection has been focusing on the use of Th1 responses elicited in immune individuals by defined parasite antigens as indicators of protection. A number of leishmanial proteins have been characterized and evidences from studies in animal models indicate that variable levels of protection can be achieved using defined protein as vaccines [START_REF] Coler | Leish-111f, a recombinant polyprotein vaccine that protects against visceral Leishmaniasis by elicitation of CD4+ T cells[END_REF][START_REF] Kushawaha | Elongation factor-2, a Th1 stimulatory protein of Leishmania donovani, generates strong IFN-gamma and IL-12 response in cured Leishmania-infected patients/hamsters and protects hamsters against Leishmania challenge[END_REF][START_REF] Singh | Cytokine responses to novel antigens in an Indian population living in an area endemic for visceral leishmaniasis[END_REF]. However, significant differences between mice and humans immune system, in both its innate and adaptive arms exist [START_REF] Mestas | Of mice and not men: differences between mouse and human immunology[END_REF]. Very few candidates have progressed beyond the experimental stage and designing an effective vaccine against leishmaniasis is still a matter of research. One of the candidate vaccine antigens is the PSA which belongs to a unique Leishmania family of membrane-bound and secreted proteins. PSA main signature is a specific Leucin Rich Repeats known to be involved in protein-protein interactions and in pathogen recognition [START_REF] Devault | The promastigote surface antigen gene family of the Leishmania parasite: differential evolution by positive selection and recombination[END_REF]. It is an abundant glycolipid-anchored protein on the surface of the promastigote form of most Leishmania species [START_REF] Devault | The promastigote surface antigen gene family of the Leishmania parasite: differential evolution by positive selection and recombination[END_REF][START_REF] Murray | The PSA-2 glycoprotein complex of Leishmania major is a glycosylphosphatidylinositol-linked promastigote surface antigen[END_REF][START_REF] Lohman | Molecular cloning and characterization of the immunologically protective surface glycoprotein GP46/ M-2 of Leishmania amazonensis[END_REF][START_REF] Hanekamp | Molecular karyotype and chromosomal localization of genes encoding two major surface glycoproteins, gp63 and gp46/M2, hsp70, and beta-tubulin in cloned strains of several Leishmania species[END_REF][START_REF] Murray | Variants of a Leishmania surface antigen derived from a multigenic family[END_REF][START_REF] Symons | Characterization of a polymorphic family of integral membrane proteins in promastigotes of different Leishmania species[END_REF][START_REF] Beetham | Glycoprotein 46 mRNA abundance is post-transcriptionally regulated during development of Leishmania chagasi promastigotes to an infectious form[END_REF][START_REF] Jimenez-Ruiz | Cloning, sequencing, and expression of the PSA genes from Leishmania infantum[END_REF]. A watersoluble form released in promastigote culture supernatants of L. major [START_REF] Symons | Characterization of a polymorphic family of integral membrane proteins in promastigotes of different Leishmania species[END_REF] and of L. infantum [START_REF] Jimenez-Ruiz | Cloning, sequencing, and expression of the PSA genes from Leishmania infantum[END_REF] has also been described. PSA is involved in parasite attachment and invasion of macrophages [START_REF] Kedzierski | A leucine-rich repeat motif of Leishmania parasite surface antigen 2 binds to macrophages through the complement receptor 3[END_REF]. It has been reported to induce Th1-mediated protection against murine leishmaniasis when used as a vaccine [START_REF] Mcmahon-Pratt | Recombinant vaccinia viruses expressing GP46/M-2 protect against Leishmania infection[END_REF][START_REF] Handman | Protective vaccination with promastigote surface antigen 2 from Leishmania major is mediated by a TH1 type of immune response[END_REF]. PSA is also specifically recognized by Th1 cells of humans with a history of self-healing CL [START_REF] Kemp | The Leishmania promastigote surface antigen-2 (PSA-2) is specifically recognised by Th1 cells in humans with naturally acquired immunity to L. major[END_REF] and by human sera of VL patients [START_REF] Boceta | Leucine rich repeats are the main epitopes in Leishmania infantum PSA during canine and human visceral leishmaniasis[END_REF]. This protein is present in both promastigote and amastigote stages [START_REF] Beetham | Surface glycoprotein PSA (GP46) expression during short-and long-term culture of Leishmania chagasi[END_REF][START_REF] Handman | The Leishmania promastigote surface antigen 2 complex is differentially expressed during the parasite life cycle[END_REF] and the presence of PSA family was demonstrated in several Leishmania species [START_REF] Devault | The promastigote surface antigen gene family of the Leishmania parasite: differential evolution by positive selection and recombination[END_REF][START_REF] Lohman | Molecular cloning and characterization of the immunologically protective surface glycoprotein GP46/ M-2 of Leishmania amazonensis[END_REF][START_REF] Mcmahon-Pratt | Loss of the GP46/M-2 surface membrane glycoprotein gene family in the Leishmania braziliensis complex[END_REF][START_REF] Myung | Comparison of the post-transcriptional regulation of the mRNAs for the surface proteins PSA (GP46) and MSP (GP63) of Leishmania chagasi[END_REF]. PSA proteins are strongly over-expressed in metacyclic promastigotes suggesting an association with the virulence status of the parasites [START_REF] Beetham | Surface glycoprotein PSA (GP46) expression during short-and long-term culture of Leishmania chagasi[END_REF]. The known role of PSA proteins in resistance to complement lysis further supports this hypothesis [START_REF] Lincoln | Genetic complementation of Leishmania deficient in PSA (GP46) restores their resistance to lysis by complement[END_REF]. The interest for the PSA is also supported by our previous [START_REF] Lemesre | Protection against experimental visceral leishmaniasis infection in dogs immunized with purified excreted secreted antigens of Leishmania infantum promastigotes[END_REF][START_REF] Lemesre | Long-lasting protection against canine visceral leishmaniasis using the LiESAp-MDP vaccine in endemic areas of France: double-blind randomised efficacy field trial[END_REF] and recent studies (unpublished data) showing that sera of dogs vaccinated with L. infantum excreted/secreted antigens (ESA) in combination with MDP (muramyl dipeptide), recognized immunodominant antigens belonging to PSA protein family, from L. infantum ESA (LiPSA-54S; GenBank under Accession number: FJ974055) and from L. amazonensis ESA (LaPSA-38S; GenBank accession number: FJ974054). In the present study, the recombinant native form of L. amazonensis PSA protein (LaPSA-38S), produced in a L. tarentolae expression system was evaluated for its ability to induce cellular and humoral immune responses. We took advantage of a multidisciplinary consortium to assess, using the same tools and samples from individuals with acquired immunity to L. major, L. braziliensis, L. infantum or L. donovani, for PSA immunogenicity and its potential use as a vaccine candidate against the different forms of leishmaniasis.

Materials and Methods

Ethics statement

The recruitment and sampling collection of different groups of volunteers, was done in accordance with Good Clinical Practice (GCP), based on the recommendations and approval of the local ethical Committee of each Institute involved in this study (Institut de Recherche pour le De ´veloppement (IRD) for France (the recruitment of blood donors and the sample collection were performed at the EFs human blood bank of Bordeaux), Instituto de Salud Carlos III (ISCIII) for Spain (Comite ´de e ´tica de la investigacion y de bienestar animal), National Institute of Pathology (NIP) for India (Office of ethical committee VMMC & SAFDARJANG hospital New Delhi and Indian council of medical research), Instituto de Medicina Tropical ''Alexander von Humboldt'' (IMTAvH) for Peru (comite ´de investigacion y e ´tica de la unidad de capacitacion) and Institut Pasteur de Tunis (IPT) for Tunisia (Comite ´d'e ´thique de l'Institut Pasteur de Tunis). A written informed consent was obtained from all subjects involved in this study.

Study population and samples

Well-characterized endemic foci, blood banks and medical structures (hospitals, medical departments and public health centers) were identified in each country (India, Peru, Spain, France, and Tunisia). Human groups (cured individuals and healthy individuals with no history of leishmaniasis but with a probable asymptomatic infection and therefore considered as immune) were recruited from endemic areas for VL and CL, based on the following defined inclusion criteria (i) living in endemic foci to L. major, L. infantum, L. braziliensis or L. donovani (ii) well documented medical records for cured groups (iii) presence of typical scars for cured CL group and (iv) high IFN-c response to Soluble Leishmania Antigens (SLA) (.300 pg/ml) for healthy individuals with a probable asymptomatic infection. Patients with active CL or VL were identified in health structures in charge of patient management and had specific clinical symptoms. Healthy individuals recruited in low or non-endemic areas, or in blood banks with no history of leishmaniasis and no or low IFN-c response to SLA (,100 pg/ml) were considered as non immune/ naives. Exclusion criteria were immunosuppressive diseases other than leishmaniasis, long term treatment and pregnancy. The different human groups used in this study are detailed in table 1. Heparinized blood was collected from a total of 104 donors and 82 controls (Table 1).

Production and purification of LaPSA-38S recombinant protein

Genomic DNA of L. amazonensis (MHOM/BR/76/LTB-012/ clone 1), cultivated in RPMI 1640 medium (Gibco-BRL, UK) and supplemented with 10% heat-inactivated FBS (Gibco-BRL, UK), 10 mM HEPES, 100 U/ml penicillin, and 100 mg/ml streptomycin (Gibco-BRL, UK) at 26uC, was extracted by phenol: chloroform: isoamyl alcohol extraction (1:1:1) and ethanol precipitation. The LaPSA-38S gene (GenBank accession number: FJ974054) was amplified with forward primer, F-PSA-38S (59-CCATGGCGCAGTGCGTGCGTCGG-39) and reverse primer, R-PSA-38S (59-GCGGCCGCGTGATGGTGATGGTGAT-GATCGTGGTTCGCCAG-39), containing NcoI and NotI restriction sites in each 59 end (underlined) by PCR reaction. The purified PCR product was cloned in pCR2.1-TOPO TA vector using TOPO TA cloning Kit (Invitrogen) following the manufacturer's procedures. The transformed cells E. coli TOP10 (Invitrogen) were screened for the presence of recombinant plasmid with the LaPSA-38S insert by gene-specific PCR and analysis with NcoIand NotIrestriction enzymes and isolated positive clones were sequenced. The insert was removed by NcoIand NotI digestion and subcloned into the NcoI and NotI insertion site of Leishmania expression vector pF4X1.4sat1 allowing selection with the antibiotic Nourseothricin (Jena Bioscience, Germany) to create the recombinant pF4X1.4-LaPSA-38S plasmid. The resulting construct, encoding a full sequence of LaPSA-38S secreted protein fused to a C-terminus His 6 tag. L. tarentolae promastigotes were successfully maintained in continuous culture by successive passages of 5610 5 flagellates/ml every week into 10 ml of completely chemically defined medium (CDM/LP) free of serum, macromolecules, proteins and cell contaminants as previously described [START_REF] Lemesre | Methods for the culture in vitro of different stages of tissue parasites[END_REF][START_REF] Merlen | Leishmania spp: completely defined medium without serum and macromolecules (CDM/LP) for the continuous in vitro cultivation of infective promastigote forms[END_REF]. For stable integration of the expression cassette into the 18S ribosomal RNA (ssu) locus, 10 mg of pF4X1.4sat1 plasmid containing LaPSA-38S gene was digested by SwaI restriction enzyme. Transfections of L. tarentolae promastigotes were performed by electroporation in 2 mm cuvettes using a Gene Pulser II (Biorad) and a single pulse (5-6 msec) with the settings 450 V and 450 mF. After pulse, cells were transferred into a fresh CDM/LP medium. 100 mg/ml of Nourseothricin was added 24 h after electroporation to select stable transformants. After a week of culture only nourseothricin-resistant cells survived. Transgenic cells were selected as single colonies on the supplemented CDM/ LP-agar medium containing 100 mg/ml Nourseothricin. To confirm the integration of the LaPSA-38S containing cassette into the Leishmania genome, PCR was performed on the genomic DNA of wild type and transgenic cells with ssu forward primer F3001 and aprt reverse primer A1715 (Jena bioscience, Germany). Mass culture and protein purification were manufactured by Virbac in GMP conditions. Culture amplifications were performed on CDM/LP medium. The culture supernatant containing L. amazonensis excreted secreted LaPSA-38S, released by the parasite during its growth, was recovered at the late stationary phase of growth. The only proteins contained in the medium were of parasitic origin and were found with their native conformation because they were naturally excreted-secreted by Leishmania parasites. Excreted secreted PSA was purified from concentrated culture supernatant by Ni-NTA affinity chromatography.

Preparation of total soluble Leishmania promastigote antigens (TSLA)

All antigen extracts were prepared from promastigote stationary phase parasite cultures of L. donovani, L. braziliensis, L. major and L. infantum. Each consortium partner used local TSLA (L. major TSLA in Tunisia, L. infantum TSLA in France and Spain, L. braziliensis TSLA in Peru and L. donovani TSLA in India) and TSLA from L. donovani (TSLA Ldd8) (MHOM/IN/80/Ldd8Cl 2 L. donovani strain (Ldd8)), as a standard preparation. TSLA were obtained from washed parasites in 1x phosphate-buffered saline (PBS), centrifuged at 10006g/10 min at 4uC and supernatants were removed. The pellets were resuspended in lysis buffer (50 mM Tris/5 mM EDTA/HCl, pH7. 1 mL/1610 9 parasites), subjected to three rapid freeze/thaw cycles followed and to three pulses of 20 seconds/40W with sonicator. Samples were centrifuged at 50006g for 20 min at 4uC, and supernatants were collected, aliquoted and stored at 280uC until use. Protein quantification was performed using Bradford method.

Cell culture and stimulation

Peripheral blood mononuclear cells (PBMC) were isolated from blood by density centrifugation through Ficoll-Hypaque (GE Healthcare Bio-Sciences AB, Uppsala, Sweden).

The cells were cultured in RPMI 1640 supplemented with 10% heat inactivated FBS, 100 IU/mL penicillin, 100 mg/mL strepto- mycin, 2 mM L-glutamin, 50 mM 2-mercaptoethanol, 1 mM sodium pyruvate and 1X amino acid non essential. Briefly, cells were plated in 96 well (TPP, Switzerland) and were kept with media alone (unstimulated) or stimulated with 10 mg/mL of Phytohemagglutinin (PHA) (Sigma-Aldrich) as a positive control or with TSLA (10 mg/mL) or with LaPSA-38S (10 mg/mL), in a 5%CO 2 humidified atmosphere at 37uC for 5 days.

Cytometric Bead Array assay (CBA)

IFN-c, granzyme B, tumor necrosis factor-alpha (TNF-a) and Interleukin-10 (IL-10), were detected and quantified from culture supernatants (50 ml) of PBMC exposed for 120 h to the following stimuli: PHA-M (10 mg/ml), TSLA (10 mg/ml) or LaPSA-38S (10 mg/ml), using the BD CBA Human Soluble Protein Flex Set system and according to the instructions of the manufacturer (BD Biosciences). It should be noted that granzyme B was not analyzed in individuals immune to L. braziliensis infection. In order to quantitate samples, the BD CBA Human Soluble Protein Flex Standard was performed for each cytokine and in each experiment. Data were acquired by flow cytometry (FACSCalibur or FACSCanto) using 2-color detection. Flow Cytometric Analysis Program Array (FCAP Array; BD Biosciences) software was required for analyzing samples [START_REF] Morgan | Cytometric bead array: a multiplexed assay platform with applications in various areas of biology[END_REF].

Cell phenotyping

Freshly isolated PBMC were stimulated with PMA (50 ng/ml)/ ionomicyn (10 26 M) or PHA at 10 mg/ml for 6 h (positive control) or with TSLA at 10 mg/ml or with LaPSA-38S at 10 mg/ml for 120 h and, or kept with medium alone. Cells were treated with Golgistop (BD Biosciences) for the last 6 hours of culture, then washed and incubated with antibodies: FITC CD3, PerCPcy5.5 CD4, APC-H7 CD8 or PerCPcy5.5 CD8, and PE-Cy7 CD69 or PE CD69 (BD Biosciences), for 20 minutes at 4uC. For intracellular IFN-c detection, cells were fixed and permeabilized using BD Cytoperm/cytofix plus kit (BD Biosciences) according to manufacturer's instructions and labeled with PE-anti-IFN-c mAb (intracellular formulation) (BD Biosciences). Analysis was performed with FACSCalibur using CellQuest Pro software or FACS canto II flow cytometer using DIVA software. FITC mouse IgG1, PerCPCy5.5 mouse IgG1, mouse IgG1PE, PE mouse IgG2a (intracellular formulation) (Biosciences) were used as isotype controls for acquisition by FACSCalibur. For acquisition by FACSCanto, BD CompBeads Set Anti-Mouse Ig, k (Anti-Mouse Ig, k/Negative Control (FBS) Compensation Particles Set) (BD Biosciences) were used.

Detection of antibodies specific for LaPSA-38S

An enzyme-linked immunosorbent assay (ELISA) test was performed to detect specific antibodies to TSLA Ldd8 and LaPSA-38S in individual serum samples from the different studied groups (Table 2). In order to improve antibodies detection, ELISA protocol was optimized by combining antigens concentrations and anti-serum dilutions. Briefly, 96 wells plates (NuncMaxisorp Immuno Plates, PolyLabo, Strasbourg, France) were coated with 100 ml/well of 2 or 5 mg/ml LaPSA-38S and TSLA overnight at 4uC. Plates were washed three times with PBS, 0.1% Tween 20 (PBS-T), pH 7.4 and blocked with 200 ml/well of PBS containing 0.1% Tween 20 and 3% BSA for 1 h at 37uC. After washing with PBS-T, diluted sera (1/100, 1/150 and 1/200 in PBS-T) were added (100 ml/well) and incubated for 1h30 at 37uC. Plates were then washed with PBS-T and 100 ml/well of diluted HRPconjugated anti-human IgG in washing buffer were added for 30 min at 37uC (1/5000). Plates were developed with the 100 ml/ well of Sigma Fast o-phenylene diamine dihydrochloride (OPD) for Phenotypic analysis of LaPSA-38S and SLA-specific CD4+ and CD8+ was performed for PBMC among CD3+ population, in groups in which we observed a significant and specific LaPSA-38S-induced IFN-c response (CCLm: n = 7, HHR-Lm: n = 6, HHR-LiS: n = 7). Naive individuals (HLR-S: n = 14) were also analyzed. Data are means percentage 6 SD.

doi:10.1371/journal.pone.0092708.t002

15 min. The reaction was stopped with 50 ml/well of 2 N HCl.

The absorbance was measured at 492 nm. All sera were tested in duplicate/triplicate, and the mean value was recorded.

Statistical analysis

Data from all partners were collected into a unique database using the open source software Epidata 3.1 (Epidata Association, Denmark http://www.epidata.dk/) and checked for consistency. Individuals with missing data or exclusion criteria were deleted. Data analysis was then performed with Stata statistical software (StataCorp. 2009. Stata Statistical Software: Release 11. College Station, TX: StataCorp LP.). We used non-parametric statistical tests because of the low number of individuals in some groups and of the heterogeneity of the standard deviation between groups. Results are expressed as mean6standard deviation (SD) and a pvalue of ,0.05 was considered significant in all cases. In the statistical analysis of cytokine level and phenotyping, we used Wilcoxon signed-rank test to compare median levels of cytokines or percentage of cells producing IFN-c after different stimulations in paired data. Kruskal-Wallis rank test was used for inter-groups analysis on normalized data after deducting the non-stimulated value. Correlations were performed using pairwise correlations of variance test. For humoral response analysis, we used Kruskal-Wallis rank test to compare the median production of total IgG between different stimulations. We estimated a threshold of positivity for each country by calculating a cut-off using the following formula:

t pos ~mean level in nai if group ð Þ z 3sd level in nai if group ð Þ :
Individuals with a level of IgG after stimulation higher than t pos were considered as positive responders. For the statistical analysis of sub-classes, results were compared only for positive individuals

Results

Production of LaPSA-38S in L. tarentolae

In this study, we generated a recombinant L. tarentolae strain expressing exogenously the L. amazonensis LaPSA-38S gene. The resulting construct encoded a full sequence of LaPSA-38S secreted protein fused to a C-terminus His 6 tag. LaPSA-38S protein purity was assessed by SDS-PAGE using 12% polyacrylamide Ready Gels (Bio-Rad) under reducing and non reducing conditions and after silver staining. Immobilized Ni 2+ affinity chromatography of culture supernatant containing excreted/secreted extracts from recombinant L. tarentolae, yielded a preparation featuring a purified recombinant LaPSA-38S protein migrating as a 45 kDa species in SDS-PAGE (Figure 1). The extra band observed in figure 1 was identified as a dimer form of LaPSA-38S as suggested by a proteomic study of purified LaPSA-38S using a combination of 1D gel electrophoresis and LC-MS/MS (coupling between liquid chromatography and tandem mass spectrometry) (Bras-Gonc ¸alves et al., manuscript in preparation).

IFN-c, granzyme B, TNF-a, and IL-10 levels in response to LaPSA-38S protein in immune individuals

The supernatants of TSLA or LaPSA-38S-stimulated PBMC were assayed for IFN-c, granzyme B, TNF-a and IL-10 production (Figure 2, Figure 3). PBMC were isolated from (i) individuals cured from L. major (CCLm), L. braziliensis (CCLb) or L. donovani (CVLd) infection, (ii) healthy individuals living in endemic areas and showing high IFN-c response to TSLA, considered as immune (HHR-Lm: Healthy High Responder living in endemic area to L. major infection and HHR-Li, HHR living in endemic area to L. infantum infection) and (iii) healthy individuals living in low or non-endemic areas and showing low or no IFN-c response to TSLA, considered as non immune/naive (HLR-T: Healthy Low Responders from Tunis; HLR-F from France; HLR-S from Spain; HLR-I from India and HLR-P from Peru). HHR-Li individuals were recruited from three different areas (France: HHR-LiF; Spain: HHR-LiS; Tunisia: HHR-LiT). LaPSA-38S induced significant (when compared to non stimulated cultures, p#0.003) and specific (when compared to corresponding naive groups, p#0.01) levels of IFN-c in PBMC cultures of CCLm, HHR-Lm, HHR-LiT and HHR-LiS (Mean 6 SD: 410.96913.1 pg/ml; 297.26306.9 pg/ml; 297.26423.2 pg/ml; and 127.3679.1 pg/ml, respectively) (Figure 3a). No significant IFN-c levels were detected in HHR-LiF or in naive groups HLR-T, HLR-F or HLR-S. Low but significant IFN-c levels were observed in CCLb and CVLd but also in their related naive groups (HLR-P and HLR-I, respectively) (Figure 3a). LaPSA-38S also induced significant and specific (p#0.04) granzyme B levels in CCLm (235.76448.5), HHR-Lm (311.76345.9) and HHR-LiS (492.86451) (Figure 3b). Granzyme B was not observed in naive individuals. The levels of IFN-c and granzyme B were significantly higher in the supernatants of all immune groups (CCLm: 3027.161356. TSLA Ldd8 stimulation (Figure 2a andb). It should be noted that no significant differences were observed in IFN-c and granzyme B responses between local TSLA and TSLA Ldd8 (Figure 2a andb). Significant TNF-a levels (p#0.02) were observed in CCLb (158.56216.2), CVLd (183.96271.4) and HHR-LiS (54.7646), but also in some naive groups (HLR-P: 122.2672 and HLR-I: 253.76227.3) after LaPSA-38S stimulation (Figure 3c). TSLA Ldd8 induced significant and specific TNF-a production (p#0.03) in the same groups but also in HHR-LiT (CCLb: 149.36333.5; CVLd: 47.2650.8; HHR-LiS: 275.26152.8 and HHR-LiT: 529.26359.9) (Figure 3c). PBMC from all immune and naive groups showed significant levels of IL-10 (p#0.02) in response to LaPSA-38S (immune groups: CCLm: 218.56184.3; CCLb: 797.861110; CVLd: 74.1654.9; HHR-Lm: 233.46241.7; HHR-LiT: 104.5656.2; HHR-LiF: 87.9637.9; HHR-LiS: 175.76244.8; naive groups: HLR-T: 73.5654.2; HLR-P: 449.46432.6; HLR-I: 78.8683.6; HLR-F: 71.8638.3; HLR-S: 24.5617.6) (Figure 3d). IL-10 levels were significantly higher in CCLm, HHR-Lm and HHR-LiT compared to corresponding naive groups (HLR-T) (p# 0.009). No IL-10 was observed in response to TSLA. No difference was observed in the cytokine levels between immune groups, except for LaPSA-38S-induced granzyme B levels which were significantly higher in HHR-LiS compared to CCLm and HHR-Lm (p = 0.02 and 0.04, respectively).

Interestingly, strong positive correlations were found between IFN-c and granzyme B production in CCLm group (r = 0.82; p = 0.0000) and between granzyme B and IL-10 production in HHR-LiS group (r = 0.7; p = 0.03), in response to LaPSA-38S. IFN-c and granzyme B productions were also highly correlated in response to TSLA Ldd8 in CCLm and HHR-Li groups (r = 0.67; p = 0.0002 and r = 0.76; p = 0.01; respectively).

Phenotyping results

Phenotypic analysis of LaPSA-38S and TSLA Ldd8-specific CD4+ and CD8+ cells producing IFN-c was performed in groups in which we observed a significant LaPSA-38S-induced IFN-c response (CCLm: n = 7, HHR-Lm: n = 6, HHR-LiS: n = 7) and in naive individuals (HLR-S: n = 14) (Figure 4).

We showed a significantly higher percentage of CD4+ T cells producing IFN-c (mean +/2 SD: 0.14 +/20.21%) among activated CD4+ T cells (57.4+/213.9%), after LaPSA-38S stimulation, only in CCLm individuals, in comparison to non stimulated cultures (p = 0.049), (Figure 4, table 2). A strong positive correlation was found between the percentage of IFN-c-producing CD4+ T cells and IFN-c production in cell culture supernatants assessed by CBA analysis, in response to LaPSA-38S antigen (r = 0.97, p = 0.0003). No significant increase was observed in the percentage of IFN-c-producing CD8+ T cells in tested immune groups, in response to LaPSA-38S, compared to non stimulated cultures (Figure 4). A significant increase was showed in the percentage of IFN-c-producing CD4+ T cells in CCLm (0.37+/2 0.47%) and HHR-LiS (0.87+/20.74) and of IFN-c-producing CD8+ T cells in HHR-LiS (0.53+/20.4), compared to non stimulated cultures (p = 0.02), in response to TSLA Ldd8 stimulation (Figure 4).

Humoral response to LaPSA-38S

Anti-LaPSA-38S total IgG responses were analyzed by using ELISA tests in sera from (i) patients with active disease (aVLd, aVLiT, aVLiS, aCLb), (ii) cured individuals (CVLd, CCLm, CCLb), (iii) healthy individuals with high IFN-c response to TSLA Ldd8 as immune with no clinical symptoms (HHR-Lm, HHR-LiT, HHR-LiF, HHR-LiS) and healthy individuals with low IFN-c response to TSLA Ldd8 as naı ¨ve controls (Table 3). The cut-off value of reactivity for LaPSA-38S antigen was calculated as the mean plus 3 SD of the OD values observed in naive controls from each country. These cut-off values allowed us to distinguish between positive and negative sera and consequently to estimate performance parameters of each ELISA test (Table 3). Globally, the results indicated that LaPSA-38S antigen does not constitute a target for the humoral response in the different studied groups. The percentage of positivity was very low ranging from 0% to 12.5%. However, an exception was observed with the aVLiS group where LaPSA-38S was recognized by 50% of tested sera. In contrast and as expected, highest positivity was observed using TSLA antigen. Best results were obtained with sera from individuals with active VL diseases which 100% react with TSLA Ldd8.

Discussion

Few vaccine candidates have progressed beyond the experimental stage in animal models of leishmaniasis (mice, hamsters). This is consistent with the difficulty in developing a suitable animal model that reproduces the features of the human disease. Direct extrapolation of data from animal models to human disease is often controversial and the protective immune responses controlling experimental infection may not reflect those required to prevent leishmaniasis in endemic areas. The purpose of this study was to evaluate the ability of a PSA antigen to elicit an ex vivo cellular immune response in PBMC of non exposed and previously exposed human subjects with and without evidence of protective immunity. The rationale is that antigens that elicit immune responses correlating with protection or cure (e.g. significant IFN-c production in previously exposed and cured individuals) may be good candidates for prophylactic and/or therapeutic vaccines.

We have previously demonstrated that naturally ESA purified from culture supernatants of L. infantum promastigotes formulated with an adjuvant were able to induce a long lasting Th1-mediated protection against experimental and natural canine VL [START_REF] Devault | The promastigote surface antigen gene family of the Leishmania parasite: differential evolution by positive selection and recombination[END_REF][START_REF] Beetham | Surface glycoprotein PSA (GP46) expression during short-and long-term culture of Leishmania chagasi[END_REF][START_REF] Lemesre | Protection against experimental visceral leishmaniasis infection in dogs immunized with purified excreted secreted antigens of Leishmania infantum promastigotes[END_REF][START_REF] Lemesre | Long-lasting protection against canine visceral leishmaniasis using the LiESAp-MDP vaccine in endemic areas of France: double-blind randomised efficacy field trial[END_REF]. Interestingly, a native soluble L. infantum PSA has been identified as the active constituent of ESA that reproduce the observed protective immunity when used as a vaccine in dogs (unpublished data). PSA is present in both promastigote and amastigote stages and is over-expressed in stationary phase parasites [START_REF] Devault | The promastigote surface antigen gene family of the Leishmania parasite: differential evolution by positive selection and recombination[END_REF][START_REF] Beetham | Surface glycoprotein PSA (GP46) expression during short-and long-term culture of Leishmania chagasi[END_REF][START_REF] Handman | The Leishmania promastigote surface antigen 2 complex is differentially expressed during the parasite life cycle[END_REF]. It is highly conserved in its N-terminal and C-terminal parts, among Leishmania species, which is a requisite for ensuring cross-species protection (unpublished data). PSA isolated from L. amazonensis promastigotes as well as recombinant vaccinia viruses expressing this protein have been demonstrated to elicit a protective immune response against infection with L. amazonensis in BALB/c mice [START_REF] Mcmahon-Pratt | Recombinant vaccinia viruses expressing GP46/M-2 protect against Leishmania infection[END_REF][START_REF] Champsi | Membrane glycoprotein M-2 protects against Leishmania amazonensis infection[END_REF]. Moreover, PSA-2 polypeptides purified from L. major and plasmid DNA encoding PSA-2, but not recombinant PSA-2 purified from E. coli, provided significant protection against L. major infection in BALB/c mice [START_REF] Handman | Protective vaccination with promastigote surface antigen 2 from Leishmania major is mediated by a TH1 type of immune response[END_REF][START_REF] Sjolander | Induction of a Th1 immune response and simultaneous lack of activation of a Th2 response are required for generation of immunity to leishmaniasis[END_REF]. Our aim was to identify a human vaccine candidate targeting Leishmania species that cause CL and VL. We used a recombinant native L. amazonensis PSA (LaPSA-38S), as a potential candidate vaccine. Despite the fact that the amino sequence alignment of LaPSA-38S with PSA proteins from several Leishmania species, shows that these proteins are not 100% identical, the N-terminal and C-terminal regions present highly conserved segments. However, the number of Leucine Rich Repeat motifs located in the central region was variable [START_REF] Devault | The promastigote surface antigen gene family of the Leishmania parasite: differential evolution by positive selection and recombination[END_REF]. Nevertheless, these proteins belong to the same unique PSA family according to various well known criteria which constitute the PSA family unique signature [START_REF] Devault | The promastigote surface antigen gene family of the Leishmania parasite: differential evolution by positive selection and recombination[END_REF]. Thus, all PSA might be considered as highly similar regarding their protein primary structure since they correspond to the same architecture depicted by a precise domain organization which is well conserved among PSA from different Leishmania species [START_REF] Devault | The promastigote surface antigen gene family of the Leishmania parasite: differential evolution by positive selection and recombination[END_REF].

LaPSA-38S was produced in a non-pathogenic L. tarentolae recombinant expression system. This expression system allows the conservation of Leishmania-type post-translational modifications including glycosilation, phosphorylation and disulfide bond formation. Moreover, the L. tarentolae expression system, in addition to being non-pathogenic to mammals, is an ideal expression system to produce native proteins of Leishmania [START_REF] Breitling | Nonpathogenic trypanosomatid protozoa as a platform for protein research and production[END_REF]. A multi-disciplinary consortium has been set-up including groups from Europe (France and Spain for L. infantum infection) and from different endemic countries where both VL and CL are highly prevalent (India for L. donovani, Peru for L. braziliensis and Tunisia for L. major and L. infantum infections). We demonstrated that LaPSA-38S induced similar significant and specific levels of IFN-c in individuals immune to L. major and L. infantum infection. Healthy high IFN-c responders had equivalent responses to the cured group. A non specific IFN-c response to LaPSA-38S stimulation was observed in L. braziliensis or L. donovani immune individuals. Analysis of the phenotypes of LaPSA-38S-stimulated T cells showed a significant increase in the percentage of IFN-c producing CD4+ T cells detected only in cured individuals. Furthermore, a strong positive correlation was found between the percentage of IFN-c-producing CD4+ T cells and the IFN-c production determined by CBA. Data on the PSA immunogenicity in humans are scarce. One study has shown that PSA from L. amazonensis was able to stimulate T cell proliferation in leishmaniasis patients [START_REF] Burns | Characterization of a membrane antigen of Leishmania amazonensis that stimulates human immune responses[END_REF]. To our knowledge, only one study reported that PSA from L. major was able to induce strong proliferative responses and high IFN-c and TNF-a levels in individuals with a past history of self-healing CL [START_REF] Kemp | The Leishmania promastigote surface antigen-2 (PSA-2) is specifically recognised by Th1 cells in humans with naturally acquired immunity to L. major[END_REF]. Healing from human leishmaniasis is generally associated with an appropriate Th1 response characterized by IFN-c Figure 2. Total Soluble Leishmania antigen (TSLA) specific IFN-c, granzyme B, TNF-a responses. IFN-c (Fig. 2a), granzyme B (Fig. 2b), were detected and quantified from culture supernatants of PBMC exposed for 120h to local TSLA (10 mg/ml) and TSLA Ldd8 (10 mg/ml), by Cytokine Beads Array test (CBA) using Flow cytometry. Statistically significant differences between stimulated and non stimulated cultures and between groups (p# 0.03) are showed. doi:10.1371/journal.pone.0092708.g002 Figure 3. LaPSA-38S specific IFN-c, granzyme B, TNF-a and IL-10 responses. IFN-c (Fig. 3a), granzyme B (Fig. 3b), TNF-a (Fig. 3c) and IL-10 (Fig. 3d) were detected and quantified from culture supernatants of PBMC exposed for 120 h to SLA (10 mg/ml) or LaPSA-38S (10 mg/ml) using Cytokine Beads Array test (CBA). Data were analyzed by Flow cytometry. PHA (10 mg/ml) was used for all cultures as positive control (data not shown). Statistically significant differences between stimulated and non stimulated cultures (p#0.003) and between groups (p#0.01) are showed. doi:10.1371/journal.pone.0092708.g003

The cut-off value of reactivity for LaPSA-38S antigen was calculated as the mean plus 3 SD of the OD values observed in naive controls from each country. These cut-off values allowed us to distinguish between positive and negative sera and consequently to estimate performance parameters of each ELISA test. doi:10.1371/journal.pone.0092708.t003

production. Thus, our finding showing that LaPSA-38S stimulates high levels of IFN-c in individuals with immunity to L. major or L. infantum infection, suggests that this protein may be associated with protective immunity. With regard to IL-10 production, we showed that, in addition to its Th1 response-inducing activity, LaPSA-38S induces significant IL-10 levels in individuals with immunity to L. major or L. infantum infection, and to a lesser extent in naı ¨ve groups. IL-10 is an anti-inflammatory cytokine with broad immuneregulatory functions, produced by different cell populations including dendritic cells, macrophages, Th2 and different regulatory CD4+ T subsets [START_REF] Moore | Interleukin-10[END_REF]. The regulatory effects of IL-10 on immune responses and pathology have been frequently reported [START_REF] Jankovic | IL-10 production by CD4+ effector T cells: a mechanism for self-regulation[END_REF]. Heterogeneity of CD4+ T cell cytokine response has complicated the definition of the immune correlates of vaccinemediated protection [START_REF] Alexander | T helper1/t helper2 cells and resistance/ susceptibility to leishmania infection: is this paradigm still relevant?[END_REF]. Recently, the balance between proinflammatory IFN-c/TNF-a and regulatory IL-10 cytokines has been shown to be involved in the outcome of human leishmaniasis and in the prediction of vaccine success [START_REF] Singh | Cytokine responses to novel antigens in an Indian population living in an area endemic for visceral leishmaniasis[END_REF]. Therefore, the capacity of LaPSA-38S to induce IL-10 does not exclude this protein as a potential candidate vaccine. We also detected significant and specific levels of granzyme B in L. major and L. infantum immune individuals. Very few reports have investigated the involvement of granzyme B in human leishmaniasis. While an increase of granzyme B activity have been associated with a good prognosis in human CL caused by L. major or L. mexicana, [START_REF] Bousoffara | Analysis of granzyme B activity as a surrogate marker of Leishmania-specific cell-mediated cytotoxicity in zoonotic cutaneous leishmaniasis[END_REF][START_REF] Hernandez-Ruiz | CD8 cells of patients with diffuse cutaneous leishmaniasis display functional exhaustion: the latter is reversed, in vitro, by TLR2 agonists[END_REF], CD8+ granzyme B+ T cells have been reported to mediate tissue injury in CL caused by L. braziliensis [START_REF] Santos | CD8(+) Granzyme B(+)-Mediated Tissue Injury vs. CD4(+)IFNgamma(+ )-Mediated Parasite Killing in Human Cutaneous Leishmaniasis[END_REF]. Granzyme B is a serine protease expressed in human T CD8+ and T CD4+ cells as well as in Treg cells [START_REF] Grossman | Differential expression of granzymes A and B in human cytotoxic lymphocyte subsets and T regulatory cells[END_REF]. It has been first implicated in apoptosis of target cells [START_REF] Vignali | How regulatory T cells work[END_REF]. Recently, several studies have highlighted a new inflammatory role of granzyme B through the regulation of cytokine expression and processing and regulatory T cells activity [START_REF] Vignali | How regulatory T cells work[END_REF][START_REF] Afonina | Granzyme B-dependent proteolysis acts as a switch to enhance the proinflammatory activity of IL-1alpha[END_REF][START_REF] Hiebert | Granzyme B in injury, inflammation, and repair[END_REF][START_REF] Magnani | Killing of myeloid APCs via HLA class I, CD2 and CD226 defines a novel mechanism of suppression by human Tr1 cells[END_REF]. To our knowledge, this is the first description of a Leishmania antigen-induced granzyme B response in humans immune to Leishmania infection. The increase of granzyme B, in response to a heterologous prime boost vaccination of mice using the LACK p36 antigen suggests its contribution to the disease control [START_REF] Dondji | Intradermal NKT cell activation during DNA priming in heterologous prime-boost vaccination enhances T cell responses and protection against Leishmania[END_REF]. Our data also show strong positive correlations between granzyme B and IFN-c levels in response to LaPSA-38S, in individuals with cured L. major CL, which could be associated with protective immunity. We also showed in our study that LaPSA-38S induced a mixed Th1 and Th2/Treg cytokine profile in individuals with immunity to L. major and L. infantum infection but not in individuals with immunity to L. braziliensis or L. donovani. Data on conserved Leishmania antigens enabling cross-species protection were reported in murine models [START_REF] Aguilar-Be | Cross-protective efficacy of a prophylactic Leishmania donovani DNA vaccine against visceral and cutaneous murine leishmaniasis[END_REF][START_REF] Goto | Leishmania infantum sterol 24-c-methyltransferase formulated with MPL-SE induces crossprotection against L. major infection[END_REF][START_REF] Ramirez | Evaluation of immune responses and analysis of the effect of vaccination of the Leishmania major recombinant ribosomal proteins L3 or L5 in two different murine models of cutaneous leishmaniasis[END_REF]. However, absence of cross-species protection was more frequently described [START_REF] Melby | Leishmania donovani p36(LACK) DNA vaccine is highly immunogenic but not protective against experimental visceral leishmaniasis[END_REF][START_REF] Salay | Testing of four Leishmania vaccine candidates in a mouse model of infection with Leishmania (Viannia) braziliensis, the main causative agent of cutaneous leishmaniasis in the New World[END_REF][START_REF] Tonui | Cross-protection against Leishmania donovani but not L. Braziliensis caused by vaccination with L. Major soluble promastigote exogenous antigens in BALB/c mice[END_REF]. The absence of cross-protection between Leishmania species could be explained by the biological differences between Leishmania species and by the level of sequence homology of the Leishmania proteins used. Indeed, while the L. major and L. infantum proteins-coding sequences are conserved, the new world L. braziliensis proteins-coding sequences are more divergent [START_REF] Peacock | Comparative genomic analysis of three Leishmania species that cause diverse human disease[END_REF]. Finally, humoral responses analysis results clearly show that LaPSA-38S antigen did not induce a significant antibody response in individuals that display different clinical manifestations and forms of leishmaniasis. These results are in accordance with other studies describing reactivities of human VL sera to PSA antigen [START_REF] Beetham | Surface glycoprotein PSA (GP46) expression during short-and long-term culture of Leishmania chagasi[END_REF]. However, in our case, we cannot exclude that this low recognition may be due to the aminoacids sequence divergence between LaPSA-38S (used in our experiment) and PSA from L. major, L. infantum and L. donovani. In conclusion, we have demonstrated here that, in spite of differences in disease forms induced by L. major and L. infantum, the LaPSA-38S protein was able to induce a similar Th1 and Th2/Treg profile in individuals immune to infection by these two species. To our knowledge, this is the first description of PSA-induced IFN-c, granzyme B, TNF-a and IL-10 response in humans with immunity against L. major and L. infantum infection. The capacity of LaPSA-38S to induce mixed Th1 and Th2/T regulatory responses is an argument for the use of this protein as a potential candidate vaccine. Our approach to investigate the ability for a given antigen, to elicit cellular protective responses in human populations from different endemic areas may help to select vaccine candidates and to prioritize antigens for clinical development of a subunit vaccine against leishmaniasis.

  4 and 2852.262035.9; CCLb: 740.261145.6, granzyme B was not tested; CVLd: 740.16824.4 and 1511.161549.5; HHR-Lm: 3182.361198.4 and 2608.561818.1; HHR-LiT: 3311.86818.3 and 3873.663401.8; HHR-LiF: 778.86299.3 and 364.56377.9; HHR-LiS: 2946.363773.7 and 747865902.6; respectively) compared to naives (p#0.007) after

Figure 1 .

 1 Figure 1. SDS-PAGE and silver staining of purified recombinant La-PSA38S protein expressed in L. tarentolae. Purified La-PSA38S was separated by electrophoresis in a 12% SDS-PAGE and silver stained under reducing (R) and non reducing (NR) conditions. The arrow indicates the position of La-PSA-38S (45 kDa) and Molecular Weight markers were marked in kDa. doi:10.1371/journal.pone.0092708.g001

Figure 4 .

 4 Figure 4. Phenotype of IFN-c producing cells. PBMC were stimulated with PMA (50 ng/ml)/ionomicyn (10 26 M) or PHA (10 mg/ml) for 6 h (positive controls), TSLA Ldd8 (10 mg/ml) or LaPSA-38S (10 mg/ml) for 120 h. For intracellular IFN-c detection, cells were treated with Golgistop for the last 6 hours of culture, fixed and permeabilized using BD Cytoperm/cytofix kit, and analyzed by flow cytometry. Results represent the frequency of IFN-c producing cells within the CD3 + CD4 + CD69 + and CD3 + CD8 + CD69 + cell populations. * Statistically significant differences from stimulated and non stimulated cultures (p,0,05). doi:10.1371/journal.pone.0092708.g004

Table 1 .

 1 Study population.

	Country	Human groups*	Number of individuals	Parasite species
	India	CVLd	16	L. donovani
		aVLd	25	L. donovani
		HLR-I	19
	Peru	CCLb	8	L. braziliensis
		aCLb	21	L. braziliensis
		HLR-P	10
	Tunisia	CCLm	25	L. major
		HHR-Lm	20	L. major
		HHR-LiT	18	L. infantum
		aVLiT	12	L. infantum
		HLR-T	16
	France	HHR-LiF	8	L. infantum
		HLR-F	28
	Spain	HHR-LiS	9	L. infantum
		aVLiS	4	L. infantum
		HLR-S	9

The recruitment and sampling collection (186 donors) of different groups (patients, cured, immunes without clinical symptoms and naives) were performed in endemic and non-endemic areas in each country. *CVLd: Cured Visceral Leishmaniasis due to L. donovani (India), aVLd: active Visceral Leishmaniasis due to L. donovani (India), HLR-I: Healthy Low Responders from India, CCLb: Cured Cutaneous Leishmaniasis due to L. brasiliensis (Peru), aCLb: active Cutaneous Leishmaniasis due to L. brasiliensis (Peru), HLR-P: Healthy Low Responders from Peru, CCLm: Cured Cutaneous Leishmaniasis due to L. major (Tunisia), HHR-Lm: Healthy High Responders living in an endemic area for L. major (Tunisia), HHR-LiT: Healthy High Responders living in an endemic area for L. infantum (Tunisia), aVLiT: active Visceral Leishmaniasis due to L. infantum (Tunisia), H-T: Healthy Low Responders from Tunisia, HHR-LiF: Healthy High Responders living in an endemic area for L. infantum (France), HLR-F: Healthy Low Responders from France, HHR-LiS: Healthy High Responders living in an endemic area for L. infantum (Spain), aVLiS: active Visceral Leishmaniasis due to L. infantum (Spain), HLR-S: Healthy Low Responders from Spain. doi:10.1371/journal.pone.0092708.t001

Table 2 .

 2 Frequency of CD4+ and CD8+ T cells after SLA and LaPSA-38S stimulation. ,97 56,7566,01 51,265,01 44,8610,22 53,969,45 43,4612,98 48,966,72 39,667,93 57,7610,7 57,264,70 52,169,50 43,5611,30 57,4610,41 59,563,48 52,765,49 43,269,08 CD8+T cells 25,1566,88 2766,00 20,666,59 22,965,91 24,465,66 39,6620,82 41,6610,93 47,869,83 21,366,80 25,463,10 20,765,50 21,366,10 24,564,31 25,264,95 20,665,03 23,566,25

		HLR-S
		HHR-LiS
		HHR-Lm
	LaPSA-38S	CCLm
		HLR-S
		HHR-LiS
		HHR-Lm
	SLA	CCLm
		HLR-S
		HHR-LiS
	PMA/iono or PHA	CCLm HHR-Lm
		HLR-S
		HHR-LiS
		HHR-Lm
	Medium	Groups CCLm	CD4+T 64,465	cells

Table 3 .

 3 Total IgG prevalence against LaPSA-38S in different endemic areas of leishmaniasis.

	Endemic Area	Ig	Group of individuals	Tested sera	Percentage of positive sera (%)
					SLA	LaPSA
	India	Total IgG	CVLd	13	92,3	-
			aVLd	25	100	8
			HLR-I	9	-	-
	Peru	Total IgG	CCLb	9	-	-
			aCLb	21	4,8	-
			HLR-P	10	-	-
	Tunisia	Total IgG	CCLm	19	68,4	5,3
			HHR-Lm	20	15	5
			HHR-Li	15	40	6,7
			aVLiT	12	100	-
			HLR-T	17	5,9	5,9
	France	IgG	HHR-Li F	8	12,5	12,5
			HLR-F	28	-	3,5
	Spain	IgG	HHR-Li S	9	-	-
			HLR-S	9	-	11,11
			aVLiS	4	100

PLOS ONE | www.plosone.org

May 2014 | Volume 9 | Issue 5 | e92708

Acknowledgments

We would like to thank Ahmed Chakroun for technical assistance, Lamia Guizani-Tabbane (Laboratory of Medical Parasitology, Biotechnology and Biomolecules, Institut Pasteur de Tunis) for manuscript reading, Sophie Bonnel (IRD, France) and Marie-Alice Kong-Hap (IRD, France) for their helpful contribution in the immunological studies. We are grateful to the staff of the Regional Directorate of Public Health of Kairouan, Tunisia.

Consortium members and affiliations R. Chamakh-Ayari, W. Markikou-Ouni, K. Aoun, M. Chenik and A. Meddeb-Garnaoui: Laboratory of Medical Parasitology, Biotechnology and Biomolecules, LR11-IPT-06, Institut Pasteur de Tunis, Tunisia. Elodie Petitdidier, J. Pagniez, R. Bras-Gonc ¸alves and Jean-Loup Lemesre: Institut de Recherche pour le De ´veloppement, UMR177 IRD/ CIRAD «INTERTRYP», Montpellier, France. Narges Bahi-Jaber, Laboratory of Transmission, Control and Immunobiology of Infection, LR11-IPT-02, Institut Pasteur de Tunis, Tunisie; and recently joined UPSP EGEAL Institut Polytechnique LaSalle Beauvais, France. Faten Bel Haj Rhouma, Laboratory of Transmission, Control and Immunobiology of Infection, LR11-IPT-02, Institut Pasteur de Tunis, Tunisie. E. Carrillo and J. Moreno: WHO Collaborating Center for Leishmaniasis, Centro Nacional de Microbiologia, Instituto de Salud Carlos III, Madrid, Spain. H. Kaushal, N.S. Negi and P. Salotra: National Institute of Pathology (ICMR), Safdarjung Hospital Campus, New Delhi, India. F. Falconi-Agapito, A. Privat, M. Cruz and J. Arevalo: Instituto de Medicina Tropical ''Alexander von Humboldt,'' Universidad Peruana Cayetano Heredia (UPCH), Lima, Peru. GM. Papierok: Virbac, Carros, France. P. Torres: Centro de Transfusio ´n de la Comunidad de Madrid, Spain

This study received financial support from EU's Seventh Framework Programme (FP7) (RAPSODI project, grant agreement number 223341). URL of funder's website: http://cordis.europa.eu/fp7/home_en.html). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Author Contributions


N Papoulias Rmod

M Denker

S Ducasse

L Fabresse
email: luc.fabresse@mines-douai.fr

Reifying the Reflectogram Towards Explicit Control for Implicit Reflection

Keywords: D.3.3 [Programming Languages]: Language Constructs and Features Reflection, Intercession, Reflectogram, Explicit Control

Reflective facilities in OO languages are used both for implementing language extensions (such as AOP frameworks) and for supporting new programming tools and methodologies (such as objectcentric debugging and message-based profiling). Yet controlling the run-time behavior of these reflective facilities introduces several challenges, such as computational overhead, the possibility of metarecursion and an unclean separation of concerns between base and meta-level. In this paper we present five dimensions of meta-level control from related literature that try to remedy these problems. These dimensions are namely: temporal and spatial control, placement control, level control and identity control. We argue that the reification of the descriptive notion of the reflectogram, can unify the control of meta-level execution in all these five dimensions. We present a model for the reification of the reflectogram and validate our approach through a prototype implementation in the Pharo programming environment. Finally we detail a case-study on run-time tracing illustrating our approach.

INTRODUCTION

The notion of reflection was formally introduced to programming language literature by Brian Cantwell Smith in 1982 (by means of the programming language 3-LISP [START_REF] Cantwell | Reflection and Semantics in a Procedural Programming Language[END_REF]). In OO reflective systems, reflection is concretized using a MOP (Meta-Object Protocol) [START_REF] Kiczales | The Art of the Metaobject Protocol[END_REF]. A meta-object is a regular object that describes, reflects or defines the behavior of a notion of the language in question [START_REF] Maes | Concepts and experiments in computational reflection[END_REF]. The process of materializing a notion of a language (such as an object, a class, a context or a method) as an object inside the language itself is called reification.

Reflective facilities in OO languages [START_REF] Cazzola | Reflection and meta-level architectures: State of the art and future trends[END_REF] are used both for implementing language extensions such as AOP frameworks [START_REF] Tanter | A versatile kernel for multi-language AOP[END_REF] and for supporting new programming tools and methodologies such as object-centric debugging [START_REF] Ressia | Object-Centric Reflection[END_REF] and message based profiling [START_REF] Bergel | Counting messages as a proxy for average execution time in pharo[END_REF].

Yet controlling the run-time behavior of reflection introduces several challenges such as computational overhead [START_REF] Maes | Issues in computational reflection[END_REF], the possibility of meta-recursion [START_REF] Chiba | Avoiding confusion in metacircularity: The meta-helix[END_REF] [DSD08] and an unclean separation of concerns between base and meta-level [START_REF] Bracha | Mirrors: design principles for meta-level facilities of object-oriented programming languages[END_REF]. These problems arise mainly when implicit reflection (i.e., reflection that is activated implicitly by the interpreter on pre-defined execution events [START_REF] Maes | Issues in computational reflection[END_REF]) alters the semantics of a running process in ways that lead to excess overhead or inconsistencies.

Implicit reflection operates similarly to an Event-Condition-Action model [START_REF] Klausr | The active database management system manifesto: A rulebase of adbms features[END_REF] [START_REF] Tanter | Partial behavioral reflection: Spatial and temporal selection of reification[END_REF]. In a class-based OO language the ECA model would be depicted as shown in Figure 1. The Event (left part of Figure 1) in the case of implicit reflection is a semantic action of the underlying interpreter (e.g., read/write slot, message send, method execution etc.) while both Condition and Action can be considered as custom code snippets (such as block closures) defined by the developer. In its most general form registration of such events can take the following form (code presented in Smalltalk syntax): In essence for every invocation of an Event by the interpreter (such as a message send) if a predefined Condition for that event is met (e.g., a counting flag is set), a meta-level Action is implicitly evaluated (e.g., a message counter is incremented).

Starting from this general (albeit naive) model for implicit reflection this paper presents five dimensions of meta-level control from related literature, namely: temporal and spatial control, placement control, level control and identity control. We argue that the reification of the descriptive notion of the reflectogram [START_REF] Tanter | Partial behavioral reflection: Spatial and temporal selection of reification[END_REF] can unify the control of meta-level execution in all these five dimensions. The idea of reflectogram (seen in Figure 3) was proposed by Tanter et al. as a visual depiction of the control-flow between the base and the meta-level. Our work proposes to concretize this depiction as an explicit programming language entity. We present a model for the reification of the reflectogram and validate our approach through a prototype implementation in the Pharo programming environment. Finally we detail a case-study on unanticipated tracing showing that all five dimensions are needed in practical applications and a unified abstraction (such as the reflectogram) is warranted.

The contributions of this paper are the following:

Condition Event

MsgSend

MsgReceived IvRead • A model for the reification of the reflectogram.

• An implementation of our proposition and its validation.

The rest of this paper is organized as follows: Section 2 presents the different dimensions of meta-level control. Section 3 presents our model for the reification of the reflectogram. Section 4 illustrates a case-study on unanticipated tracing using our approach. Section 5 details the prototype implementation of our approach in Pharo. Section 6 compares related work. Finally Section 7 concludes the paper and discusses future perspectives.

DIMENSIONS OF META-CONTROL

Temporal Control

We refer to the temporal control of implicit reflection as the ability of run-time installation, activation, de-activation and removal of reflective facilities. Temporal control allows a programmer to define when a semantic event will actually be reified by controlling the time of its activation. In essence setting up meta-actions for semantic events such as the one we described on Script 1 can either be done statically (at compile or load time) or dynamically at run time. In this latter case, trivial conditions like the countingFlag of Script1 are redundant since meta-actions can be installed, enabled, disabled or removed during execution:

2

... "code whose messages will be counted"

3

MsgSend disable

Temporal control of reflective facilities at runtime can support unanticipated behavioral reflection as was first illustrated by Redmond et al. [START_REF] Redmond | Supporting unanticipated dynamic adaptation of application behaviour[END_REF] for the Iguana/J framework [START_REF] Redmond | Iguana/J: Towards a dynamic and efficient reflective architecture for java[END_REF]. Röthlisberger et al. [START_REF] Röthlisberger | Unanticipated partial behavioral reflection: Adapting applications at runtime[END_REF] further optimized this approach by supporting unanticipated partial behavioral reflection in Geppetto. Examples include the temporal control of profiling facilities at run-time to facilitate memoization and caching. Röthlisberger et al. give such an example for web-applications in [START_REF] Röthlisberger | Unanticipated partial behavioral reflection: Adapting applications at runtime[END_REF].

Spatial Control

Spatial control was introduced by Tanter et al. [START_REF] Tanter | Partial behavioral reflection: Spatial and temporal selection of reification[END_REF] to support a partial reflection scheme in Reflex. Spatial control allows a programmer to narrow the scope of implicit reflection to specific entities (objects, classes, methods etc.) and operations thus optimizing performance. In a model supporting spatial control our example from Script 1 would be written as follows: The difference here on Script 3 (line 1) with our initial example is that a specific class is targeted to be interceded rather than the whole system. Implementations of spatial control -such as the one of Reflex [TNCC03] -provide even finer control over what is reified. This is accomplished by targeting single operations in a sub-method level or even restricting reifications to specific objects over particular executions as in the case of Bifrost [START_REF] Ressia | Object-Centric Reflection[END_REF]. With spatial control unnecessary jumps to the meta-level (e.g., for classes other than SomeClass) can be avoided resulting to an execution speed-up of reflective code.

Placement Control

On the other hand placement control allows a programmer to define the relative timing of an action in relation to its semantic event as exemplified by Tanter et al. [START_REF] Tanter | Partial behavioral reflection: Spatial and temporal selection of reification[END_REF], but also in works related to method slots [START_REF] Zhuang | Method slots: Supporting methods, events, and advices by a single language construct[END_REF] and wrappers [START_REF] Brant | Wrappers to the rescue[END_REF]. For example user-defined actions can be triggered before or after a semantic event or even totally replace the default semantic action. This is shown on lines 4 & 5 of Script 4 where two different meta-actions are registered to be triggered for message sends of SomeClass. The first on line 4 is a logging meta-action registered to be triggered before the actual message send in the base-level code of SomeClass. While the second action (line 5) is our counter increment example registered to be performed after the semantic event of the message send.

Spatial, temporal and placement control can be used in a variety of contexts where partial reflection is applicable. The most prominent examples can be found in implementations of AOP frameworks such as the one of [START_REF] Tanter | A versatile kernel for multi-language AOP[END_REF].

Level Control

Level control refers to the ability of assigning different reflective behavior to different meta-levels of a reflective tower [START_REF] Wand | The Mystery of the Tower Revealed: A Non-Reflective Description of the Reflective Tower[END_REF]. Conceptually in OO languages we can say that we operate in a new "higher" meta-level whenever a new reflective action is triggered from within the meta-level itself. This process can continue indefinitely if meta-level actions are not carefully coded. In this case the problem of infinite meta-recursion occurs [START_REF] Chiba | Avoiding confusion in metacircularity: The meta-helix[END_REF].

A simple case of infinite meta-recursion illustrating the problem is given on Script 5. On lines 1 to 3 of Script 1 we register (through the message #on:do:) a callback action for the MessageReceived event (line 2) of the instance anObject (line1). Essentially we want the block closure in line 3 to be triggered every time the instance anObject receives a message. Alas on line 3 in order to increment a message counter (message #incrementMessageCounter) we send another message to the instance anObject from within the meta-level. This new message-send will re-trigger the MessageReceived event re-triggering the callback on line 3, resulting in an infinite recursion. Denker et al. [START_REF] Denker | The meta in meta-object architectures[END_REF] first proposed a level control mechanism to solve the meta-recursion problem in OO languages through the reification of the metaContext which represents the level in which a meta-jump occurs. The metaContext is an implicit entity of the metalevel, in the sense that the developer does not invoke it explicitly but rather executes code or binds meta-objects to specific meta-levels (as shown on Script 6): More recently and in another context (that of AOP) the idea of executions levels has been proposed [START_REF] Tanter | Execution levels for aspect-oriented programming[END_REF][START_REF] Tanter | Execution levels for aspect-oriented programming: Design, semantics, implementations and applications[END_REF]. These execution levels provide a concrete solution to the problem of aspect loops (the equivalence of meta-recursion in AOP) for languages such as AspectScript and AspectJ.

Besides being a solution to the meta-recursion problem, level control can prove useful in other contexts of meta-circularity. Examples include the profiling of meta-level execution itself through reflection.

Identity Control

Identity control is the ability to distinguish between the receiver of a reflective message and the targeted object of a reflective operation. This distinction was investigated by Bracha and Ungar through Mirrors [START_REF] Bracha | Mirrors: design principles for meta-level facilities of object-oriented programming languages[END_REF] but has been studied in different contexts as well [Fer89] [Caz03]. AmbientTalk [MVCT + 09] was the first mirrorbased implementation specifically targeting implicit reflection.

Identity control can prove useful in situations as the one depicted in Figure 2. In Figure 2 anObjectInpector wants to inspect the slots of a base level object. This object (aPersistentObject) supports persistency (on a file or on a database) though reflective intercession. This means that the semantics for instance variable access for aPersistentObject have changed through reflection to synchronize its state with an external data storage. Let us now assume that this was achieved by instructing the compiler to transform each read and write access of instance variables in the source code to metalevel calls. For example in this case each read access of instance variables in the class PersistentObject will be redirected to the metalevel method #instVarAt which has been overridden from Object to provide the additional functionality.

Although this change in semantics for aPersistentObject is desirable, it does not make sense in the case of anObjectInspector which wants direct access to the slots of aPersistentObject without triggering the back-end (i.e., database) logic. Bracha and Ungar suggest that for such cases reflective facilities that are decomposed from the language kernel should be used. In this example a separate read access method from the one depicted in Object»#instVarAt:and which the ObjectInspector»#instVarAt: invokes -can be used. This is the case of the #objInstVarAt: method of ObjectInspector which has read access through direct virtual-machine support to object slots. What has essentially changed here between Ob-ject»#instVarAt: and ObjectInspector»#objInstVarAt: which perform the same operation, is the identity of the receiver of the reflective action. In the first case the receiver is aPersistentObject while in the latter it is anObjectInspector. In summary identity control besides solving problems as the one we described above promotes a stricter separation of concerns between base-level and meta-level functionality.

REIFYING THE REFLECTOGRAM

The notion of reflectogram was introduced by Tanter et al. [START_REF] Tanter | Partial behavioral reflection: Spatial and temporal selection of reification[END_REF] as a conceptual illustration to describe meta-level behavior to human readers:

[...] A reflectogram illustrates the control flow between the base level and the metalevel during execution.

For example in the left part of Figure 3 we see a diagram from Tanter and al. describing spatial control and partial reflection, while in the bottom part of Figure 3 we see a depiction from the same paper of temporal control. Other researchers have used similar control-flow illustrations to describe different dimensions of metalevel behavior as the diagram we reproduce from [START_REF] Denker | The meta in meta-object architectures[END_REF] (right part of Figure 3) describing level control.

Given the reflectogram's versatility for describing meta-level behavior in this Section we propose its reification as a programmable entity of the meta-level. More precisely we propose its reification as an explicit meta-object that is passed as an argument at runtime to conditions & implicit actions invoked by the underlying execution environment. Our goal is to unify the control of meta-level execution under a single abstraction for end-users.

Reifying the Reflectogram

Our proposal (shown in Figure 4) in its more general form extends the Event-Condition-Action model of implicit reflection (depicted in Figure 1) by establishing a one to many relation of both the condition and the action with the reified reflectogram. The relationship is one to many in the sense that a single action or condition can be registered for multiple objects but upon each invocation only the reflectogram corresponding to the particular object that triggered the event will be passed as an argument. Besides holding a reference to that targetObject, the reflectogram should provide meta-information (our reifications slot) for the currently triggered event whichdepending on the implementation -can be used to parametrize conditions and actions at runtime.

Event Registration.

The reflectogram controls the spatial dimension of implicit reflection at runtime through the methods #on:when:do: and #on:for:when:do:, with the latter being either a static or a classside method (depending on whether classes in the target language are first class or not). Registering an event for a specific object can be modeled as follows: As seen on lines 4 and 5 of Script 7, conditions and actions in our model receive an argument which describes and controls the "shape" of the reflectogram for each meta-level jump of a particular object. The class-side method #on:for:when:do: is used for the initial registration of an event, while its instance-side counterparts #on:when:do: provides the same functionality from within the metalevel -as a convenience -for the specific object that triggered an event.

The Reflectogram API

The API of our model is organized into five distinct protocols corresponding to the five dimensions for meta-level control discussed in Section 2: Temporal Protocol. Methods #enable, #disable and #remove as their name suggests control the actual triggering of events from within the meta-level. Implementors can choose to provide static counter parts for convenience (such as #enableFor:, #disableFor: etc.). Spatial Protocol. Methods #on:when:do:, #on:for:when:do: control spatial selection by registering events for specific objects as it has been described above. Placement Protocol. Methods #defaultAction and #returnValue:

control the placement of meta-actions. The reflectogram can invoke the default action of the base-level from within the meta-level thus implicitly defining which meta-level statements will be executed before and which after the actual semantic event. Regardless of whether the default action has been triggered from within the meta-level the value that will be returned to the base-level can be explicitly set, thus facilitating total replacement of base-level semantics.

Level Protocol. Methods #processMetaLevel and #objectMet-aLevel return the height of the currently executing meta-level or condition as in the meta-level tower model. Process metalevel returns the process-wide meta-level height, while object meta-level returns the height of meta-levels that have been triggered due to events of the reflectogram's target object.

Identity Protocol. Finally methods #at:, #at:put: and #perform:withArgs: provide read, write (for slots) and execution reflective facilities (for message sending) for the target object. These methods are implemented separately from core reflection and their corresponding message sends are received by the reflectogram rather than the target object. This way the identity of the receiver of reflective methods is controlled as was described in Section 2.5.

A usage example of the reflectogram is depicted on Script 8 where we solve the meta-recursion problem that was described in Section 2.4 (Script 5) by explicitly controlling the meta-level execution flow: Lines 1 to 3 of Script 8 register the MessageReceived event for the instance anObject. On line 4 -as before -a trivial condition is registered checking a message-counting flag. Then on lines 5 to 11 a meta-action is registered for the MessageReceived event. On line 6 the reflectogram is explicitly disabled thus temporarily allowing message sends to be received by anObject without interception. On line 7 the message #incrementCounter is send to anObject without resulting in an infinite recursion since the reflectogram has been disabled. Then on lines 8 to 9 the value that will be returned to the base-level is set to the default semantic action for MessageReceived events. This default action corresponds to the evaluation of whichever message-send (received by anObject) was intercepted and triggered the meta-jump. Finally on line 10 the reflectogram is re-enabled before returning control to the base-level, as to be able to intercept further message sends to anObject.

THE REFLECTOGRAM IN ACTION

This section presents the implementation of a non-trivial tracing framework where the code that will be traced is not a priori known (i.e., is unanticipated) but is being instrumented on-the-fly at runtime. Message-based profiling [START_REF] Bergel | Counting messages as a proxy for average execution time in pharo[END_REF] for example uses such a tracing approach to approximate execution time of selected methods. Through this example, we aim to show that all five dimensions of control co-occur in practical applications and a unified abstraction (such as the reflectogram) is warranted.

Figure 5 shows the core classes of our tracing framework which include:

CallGraph. The entry point of the output callgraph of our tracing process.

CallGraphNode. Individual nodes of the output callgraph holding the actual meta-information that have been traced. For our framework these meta-information include: the receiver of a message-send, its class, the selector and the arguments passed along with the message call.

ExecutionTrace. Users subclass ExecutionTrace adding the entry point symbol of the code to be traced by invoking the inherited #run: aSymbol method (where aSymbol corresponds to a method-name). Also inherited are the corresponding output callgraph and the process (i.e., green thread) where the tracing of a targeted method will take place.

CallTrace. Finally CallTrace implements the condition and send callbacks (Script 9) which are bound to traced objects at runtime. These callbacks then delegate meta-level control to methods #inTracingScope:, #addGraphNode:, #executeNode: and #return: respectively (Script 10). As seen in lines 2,8,9 and 10 of Script 9 since the reflectogram is reified as a first class entity it can be passed as an argument beyond the scope of conditions or meta-actions themselves. In line 2 the reflectogram is passed to #inTracingScope: method to determine if the meta-event that triggered the conditionCallback happened inside our tracing process or not. In line 8 it is passed to #addGraphNode: to gather the meta-information needed to update the callgraph. Then on line 9 it is passed to #executeNode: to perform the default baselevel action and gather its return value. Finally on line 10 it is passed to the return: method which resets both the callgraph and the reflectogram appropriately for further processing. Method #inTracingScope: (lines 1 to 4) the level protocol of the reflectogram is used (line 4) in order to determine whether we are intercepting a method call that originated from our tracing process' base-level (processMetaLevel = 1). If not #inTracing-Process: will return false and the corresponding meta-action (lines 5 through 10 on Script 9) will not be invoked. Method #addGraphNode: (lines 5 through 14) the reification slot of the reflectogram is used in order to gather meta-information about the intercepted call and update the callgraph. Method calls are intercepted every time a message is sent to a new receiver (from within a traced object). On lines 8 through 11 the identity protocol is used in order to extract the class of this new receiver and avoid the meta-recursion problem in case this receiver was previously being traced. Method #executeNode: (lines 15 to 27) a new call trace is being created and is being assigned to the new receiver at run-time via the spatial protocol (lines 19 to 23) then on lines 24 to 26 the placement protocol is being used to perform the default base-level action and gather its return value. Since the baselevel action is a method call to a newly traced object it will re-trigger the meta-level for all new method calls from within its scope before returning.

Method #return: (lines 28 to 30) is the equivalent of a post-action, we update the callgraph (to point to the node that we have previously added) and re-enable the reflectogram (line 30) for our traced object through the temporal protocol. The reflectogram had been previously disabled for convenience (in order to avoid unnecessary meta-jumps) on the beginning of the meta-action callback (line 7, Script 9).

IMPLEMENTATION

The prototype implementation of our model for the reflectogram (as described in Section 3) as well as the case-study (detailed on Section 4) are part of a dedicated virtual machine targeting the Pharo platform: the metaStackVM1 [START_REF] Papoulias | Remote Debugging and Reflection in Resource Constrained Devices[END_REF]. We chose to implement our prototype through a virtual-machine extension (rather through some other form of instrumentation, for e.g., byte-code instrumentation) since vm-support serves better the instrumentation needs of run-time entities (for e.g., terminal instances, as opposed to static entities such as classes or methods).

In order to evaluate our solution we performed a micro-benchmark to compare the overhead introduced to normal execution with and without the reflectogram reification. The benchmark is based on Tanter [START_REF] Tanter | Partial behavioral reflection: Spatial and temporal selection of reification[END_REF] and measures the slowdown introduced for one million messages sent to a test object when a) no instrumentation is present b) instrumentation is loaded but is disabled for this specific object c) instrumentation is enabled on the test object d) instrumentation is enabled on the test object and its reflectogram is being reified.

As we see in Table 1 when instrumentation is loaded to the environment but the benchmark object is not being instrumented, there is no additional overhead compared to the standard VM (with no instrumentation). This is important for practical reasons so as to avoid slowing down the whole system when instrumenting only a part of it [START_REF] Tanter | Partial behavioral reflection: Spatial and temporal selection of reification[END_REF]. For example implicit reflection on the metaStackVM introduces a 9.27x overhead but only for the benchmarked object, outperforming other solutions for the same platform [START_REF] Papoulias | Remote Debugging and Reflection in Resource Constrained Devices[END_REF]. Finally adding the reification of the reflectogram to the metaStackVM introduces a 1.37x slowdown compared to implicit reflection without such reification. We believe that the added benefit of fine-grained meta-level control using the reflectogram outweighs this additional slowdown especially when it is only introduced for objects being instrumented.

SLOWDOWN

RELATED WORK -COMPARISON

In Section 2 we presented five dimensions of meta-control that have been previously treated separately in literature. Table 2 summarizes the facilities of their corresponding implementations and compares them with our own reflectogram solution on top of the metaStackVM.

While Iguana/J [RC00] [RC02] was the first to introduce unanticipated changes (temporal control) and spatial control for the Java Aspect-Oriented Programming.

Iguana/J Reflex Gepetto Gepetto-Ext AmbientTalk Bifrost MetaStackVM [RC00] [RC02] [TNCC03] [RDT07] [DSD08] [MVCT + 09] [Res12] (Reflectogram) Temporal (partially) Spatial Placement × × Level × × × × × Identity × × × × ×
As discussed in Section 2 the problems we presented in this paper have direct analogies to issues presented in AOP literature. In the context of AOP the dimensions of spatial, temporal and placement control are embedded in the abstractions of aspects, advices and join points. Moreover the recent proposal of executions levels [START_REF] Tanter | Execution levels for aspect-oriented programming[END_REF][START_REF] Tanter | Execution levels for aspect-oriented programming: Design, semantics, implementations and applications[END_REF] solves the equivalent problem of meta-recursion by avoiding aspects loops.

Limitations.

From a model perspective our solution presents some limitations compared to Reflex or Gepetto's model which are focused on extensibility. These models introduce abstractions (such as links & hooksets) apart from that of meta-objects in order to provide a stricter separation of concerns between handling of events (hookset responsibility) and meta-level delegation (link responsibility). Other solutions such as Bifrost provide additional support for compound meta-objects allowing for composition of meta-behavior. Our approach presents a single unifying entity (the reflectogram) for meta-level control aiming at explicit handling of control-flow from within the meta-level itself.

From this perspective the reflectogram reification is more appropriate as an end-user abstraction rather than an implementor's abstraction since it does not focus on extensibility or composition. On the other hand the reflectogram is described through the Event-Condition-Action model which all implicit reflection schemes (including Bifrost, Gepetto and Reflex) share and can thus be implemented as an extension on top of them.

CONCLUSION

Our work presents five different dimensions of meta-control for implicit reflection that have been treated separately in literature, namely: temporal and spatial control, placement control, level control and identity control. It proposes a model for the reification of a previously descriptive notion -that of the reflectogram [START_REF] Tanter | Partial behavioral reflection: Spatial and temporal selection of reification[END_REF] -arguing that such reification can unify the control of meta-level execution in all five dimensions. We presented a prototype implementation of this reification in the Pharo programming environment and validated our approach through a case-study on unanticipated tracing. In terms of future work -apart from our own prototypewe would like to implement our model as an extention to other implicit reflection frameworks (such as Gepetto) and provide a formal semantic representation of the reflectogram.

Figure 1 :

 1 Figure 1: Implicit Reflection as an Event-Condition-Action model

Script 2 :

 2 Temporal Control Example 1 MsgSend do: [ messageCounter := messageCounter + 1 ].

  Transcript show: 'A message was send from SomeClass' ] 5 after: [ messageCounter := messageCounter + 1 ]

Script 5 :

 5 The meta-recursion problem

Script 6 :

 6 Code execution with a metaContext [DSD08] 1 [ ... code executing on meta-1 ... ] valueWithMetaContext 2

Figure 2 :

 2 Figure 2: Inspecting an intercessed object

Figure 3 :

 3 Figure 3: Diagram of the reflectogram in literature: Tanter and al. (left & bottom) [TNCC03] & Denker [DSD08] (right)

Script 8 :

 8 Solving the meta-recursion problem with the reflectogram

Figure 4 :

 4 Figure 4: Our proposal: Reifying the Reflectogram

Figure 5 :

 5 Figure 5: Core classes of our tracing framework

Script 9 :

 9 Tracing Callbacksself return: reflectogram ] On Script 10 we see these delegate methods in more detail: Script 10: Meta-control methods using the Reflectogram 1

Table 1 :

 1 Instrumentation Benchmark

	No instrumentation	1x
	Disabled instrumentation	1x
	Enabled instrumentation	9.27x
	Reflectogram Reification	12.71x
	(With / Without) Reflectogram	1.37x

Table 2 :

 2 Comparison in terms of Meta-Control Facilities platform, it was Reflex[START_REF] Tanter | Partial behavioral reflection: Spatial and temporal selection of reification[END_REF] that introduced partial behavioral reflection to literature (supporting spatial, temporal and placement control). On the other hand due to implementation constrains Reflex did not allow for dynamic definition of meta-level behavior at run-time as did Iguana/J. These three types of control (placement, spatial & temporal) were first available during run-time in Gepetto for Smalltalk[START_REF] Röthlisberger | Unanticipated partial behavioral reflection[END_REF]. With later extensions to the Gepetto implementation covering level control[START_REF] Denker | The meta in meta-object architectures[END_REF]. Bifrost added an object-centric model to run-time reflection expanding spatial control to execution runs[START_REF] Ressia | Object-Centric Reflection[END_REF]. AmbientTalk [MVCT + 09] was the first mirror-based implementation specifically targeting implicit reflection and has support for temporal, spatial and identity control.Our own implementation manages to cover all five dimension of meta-control through the reflectogram reification. It is mainly comparable with Gepetto[START_REF] Röthlisberger | Unanticipated partial behavioral reflection[END_REF] taking into account its later extension for level control[START_REF] Denker | The meta in meta-object architectures[END_REF].

http://ss3.gemstone.com/ss/mSVM.html

ACKNOWLEDGMENTS

The authors would like to thank Prof. Eric Tanter for his valuable feedback regarding related literature in the context of AOP and for helping us strengthen our presentation. This work was partially sponsored by the European Smalltalk Users Group: http://esug.org.