A total of 59 subjects have been enrolled in the first part of this study consisting on the optimization of Grz B test. Subjects were categorized on four groups basing on clinical, epidemiological and immunological criteria:

15 patients with active ZCL (number of ZCL lesion ranging from 1 to 7) living in the endemic region of Kairouan. All of these patients showed a positive LST reactivity.

The second part consists on a prospective study carried out in two different endemic foci for L. major (Zymodeme MON-25) infection in Tunisia: an old endemic focus (OEF) situated in the district of El Gtar, governorate of Gafsa (south west Tunisia); and a new endemic focus (NEF) situated in the district of Souk Ejjdid, governorate of Sidi Bouzid (Central Tunisia). This study was carried out as part of the research project “Advances in epidemic parameters of ZCL to validate tools for surveillance and control.” At enrolment of the study population between April and May 2001, before the parasite transmission season, the donors had a clinical examination looking for active ZCL lesions or typical scars, a blood sampling and an intradermal administration of leishmanin for LST reactivity. Repeated clinical examinations were performed during the season of emergence of lesions (between September 2001 and April 2002) to evaluate the clinical course of L. major infection, occurring after the transmission season (Figure 1). The protocols were approved by the institutional review board as detailed below. A total of 453 subjects were enrolled in this study (age, mean ± SD: 7.82 ± 1.9 years [range, 3–15 years]; male: female ratio, 0.96). The study population was selected from four primary schools in the district of El Gtar (n = 212) and from three primary schools in the district of Souk Ejjdid (n = 187). Children <6 years old and therefore, of pre-school age, from the region of souk Ejjdid were also recruited (n = 54; Table 2).

Before starting the study, written consent were obtained from participants or from their parents in case of children. Subjects enrolled in this study were verbally informed of the nature of the research project and written consent was obtained for the clinical follow-up, leishmanin skin test, and blood sampling. Consent was prepared in the native language (Arabic). The protocol was approved by the Bio-Medical Ethics Committee (BMEC) of Pasteur Institute of Tunis. The leishmanin test applied to the study population is made of leishmanin approved by WHO norms and regulation. It was previously used for thousands of individuals worldwide without significant hazards.

LST was performed by intradermal injection of 100 μL of leishmanin suspension as previously described by Sassi et al. (1999). An induration's diameter ≥5 mm indicates a positive LST reactivity.

In the present study, we used L. major (zymodeme MON25; MHOM/TN/94/GLC94) obtained from a human ZCL lesion (Kébaïer et al., 2001). Parasites were cultivated at 26°C in RPMI 1640 medium (Sigma, St. Louis, MO) containing 2 mM L-glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin, and 10% heat-inactivated fetal calf serum. Stationary-phase promastigotes were used for preparation of soluble Leishmania antigens (SLA) as previously described (Sassi et al., 1999) and for stimulation of PBMCs.

PBMCs were separated from heparinized blood samples using Ficoll-Paque (GE Healthcare) density gradient centrifugation. PBMCs were cultured in 96-well plates at a concentration of 1 × 10⁶ cells/mL in a final volume of 200 μL of RPMI 1640 medium (Sigma) supplemented with 2 mM L-glutamine (Sigma), 100 U/mL penicillin (Sigma), 100 μg/mL streptomycin (Sigma), and 10% heat-inactivated human AB serum (Sigma). PBMCs were stimulated with SLA (10 μg/mL) for 5 days and lymphoproliferative response was evaluated after adding 1μCi/well of [³H]-thymidine (Amersham, France) for the last 6 h using a liquid scintillation counter (Rack Beta, LKB Wallace, Australia). Results were expressed as Δcpm, difference between mean counts of triplicates in SLA-stimulated PBMC and mean counts of triplicates in unstimulated PBMC.

Measurement of Grz B levels was performed using ELISA described by Spaeny-Dekking et al. (1998). The Grz B captured by an anti-Grz B monoclonal antibody (MoAb GB11), fixed on microtiter plate was revealed by a biotin-labeled anti-Grz B monoclonal antibody (GB10 MoAb). Recombinant Grz B of known concentration was used for tracing a standard curve. Briefly, wells of the microtiter plates (Maxisorp, NUC) were coated overnight at 4°C with 100 μl/well of MoAb GB11 diluted on carbonate buffer, 0.1 M NaHCO₃ pH 9.6, at 2 μg/ml. Plates were subsequently washed five times with PBS containing 0.02% Tween 20. The free sites were saturated by incubation for 45 min at room temperature (RT) with 150 μl/well of PBS containing 2% tween 20. The recombinant Grz B and the samples, diluted in HPE (high performance ELISA buffer, Central laboratory of the Red Cross Blood Transfusion Service) and 100 μl were added and incubated for 1 h at RT. After washing, 100 μL of GB10 MoAb diluted at a concentration of 0.5 μg/mL in HPE buffer containing 1% (v/v) of normal murine serum (NMS). After incubation for 1 h, the wells were washed and then incubated for 20 min with 100 μl of streptavidin coupled to peroxidase (Amersham, Saclay, France) diluted at 1/10,000. Five washes were carried out before adding 100 μl/well of TMB solution (3,3′, 5,5′ tetramethylbenzidine, Merck, Darmstadt, Germany) at a concentration of 100 μg/mL containing 0.003% (v/v) H₂O₂, diluted in 0.11M sodium acetate buffer pH 5.5. After stopping the reaction with 2N H ₂SO₄, optical density was measured using an automated plate reader (Titerteck Multiscan) at 450 nm/540 nm. Grz B levels were determined by reference to the standard curve and results were expressed in pg/mL. Detection threshold was fixed at 1.42 pg/mL.

Measurement of IFN-γ level within supernatant was carried out using OptEIA set ELISA (BD Biosciences, San Jose, CA) according to the manufacturer's recommendations. The results were interpolated from the standard curve established using recombinant IFN-γ and expressed in pg/mL. Detection threshold was fixed at 6.02 pg/mL.

Statistical analysis was performed using SPSS (version 20.0; SPSS) software. The association between two parameters was assessed by the Spearman's Rank Correlation Coefficient test. The correlation is considered statistically significant if r > 0.3 with a significance level p < 0.05.

The optimal and recommended cut-off points (where applicable) were used to calculate the numbers of subject positive and negative for each test. The comparison between two groups for a studied parameter was carried out by Student's t-test (for the cohort study) or by the Mann-Witney U-test (in the validation step).

For the prospective study, further analysis was performed, including the χ2 tests applied to analyze the associations between the categorical results (LST reactivity/Scar; LST/Grz B; Scar/Grz B). Odds ratios (OR) were then computed to determine the nature of the association between different parameters by stratifying subjects as described by van Belle et al. (2004). A Fisher's exact P-value was also obtained for each association. A positive association is indicated by an odds ratio >1 (significant when p < 0.05 and the 95% confidence limits both exceed 1). Odds ratios significantly <1 indicate a negative association. Estimation of the efficacy of the immune status of the host on the protection against the development of the ZCL was performed through the calculation of the relative risk (RR) and then the preventive fraction (Rothman et al., 2008). In our case, the analyzed factors consist of positive LST reactivity, presence of typical ZCL scar and high levels of Grz B (>2 ng/ml).

Before starting the prospective study, we optimized the conditions for the analysis of Leishmania-specific cytotoxic immune response. This was done by measurement of Grz B level in supernatants of PBMCs stimulated for 5 days with promastigotes of L. major at a ratio of 3 parasites per cell. The analysis was carried out for 15 subjects with active ZCL, 24 healed ZCL subjects selected basing on the presence of typical scar and positive reactivity to LST (Scar⁺LST⁺), and a total of 20 apparently healthy donors. The latter group includes 11 subjects living in endemic areas with no history of ZCL and negative LST (Scar⁻LST⁻), and 9 subjects living outside these area (Table 1). As shown in Figure 2A, the levels of Grz B detected within supernatants of PBMC from subjects cured of the disease (mean ± SD: 20693.9 ± 19576.8 pg/mL) were significantly higher compared to those measured in ZCL patients (mean ± SD: 779.62 ± 1231.48 pg/mL; p < 0.001). In addition, levels of Grz B measured in culture supernatants of cells from apparently healthy and naïve (Scar⁻LST⁻) individuals but living in endemic region of L. major transmission were significantly higher (mean ± SD: 7088 ± 8837.11 pg/mL) compared to those detected in the supernatants of PBMCs from healthy and naïve individuals living outside endemic areas (mean ± SD: 305.06 ± 589.67 pg/mL; p = 0.002). This result is very important and may suggest that in endemic area, even in the absence of LST reactivity other marker of specific immune response (like Grz B production) could indicate a previous contact with the Leishmania parasite.

We thus used the results obtained with healthy and naïve individuals living outside endemic areas as reference for the definition of the cut-off of positivity for Grz B levels and calculated as the mean + 3 times the standard deviation of the Grz B levels produced by PBMCs from healthy negative controls living outside endemic area. According to the cut-off, fixed at 2,000 pg/ml, we found that three among the 15 subjects with active ZCL, 19 among the 24 subjects with a history of ZCL and five among the 11 healthy individuals (Scar⁻LST⁻) living in endemic area were Grz B⁺. However, none of the healthy negative controls living outside endemic areas had Grz B levels greater than the cut-off (Figure 2B).

Otherwise, a positive correlation was found between Grz B levels and those of IFN-γ measured in culture supernatants (Spearman rank correlation coefficient r = 0.583, p = 0.001; Figure 2C). Furthermore, a concordance of 61.36 and 45.09% was found between Grz B levels and results of LST and SLA-specific lymphoproliferation, respectively (Figures 2D,2E).

In a next step, we evaluated the Leishmania-specific cytotoxic immune response at the baseline (before the transmission season of the parasite) within donors included in the prospective study (n = 453). Results of the clinical examination as well as those of the LST collected at the baseline showed that 167 subjects had a positive LST, among them 75 individuals exhibited ZCL scars (Table 2). Among the remaining individuals with a negative LST, 30 individuals showed ZCL scars. Interestingly, the percentage of individuals with ZCL scars is higher in the old focus (28%; 60/212) compared to the new one (18.7%; 45/240). In addition, similar percentages of subjects with positive LST reactivity were observed in both of focus (33.9% in OEF and 39.5% in NEF). However, in the NEF, this percentage is lower within subjects under 6 years old (27%) compared to that of subjects older than 6 years (43%) indicating a limited contact with the parasite for this age group.

Globally, Grz B was detected within 72.18% (327/453) of culture supernatants at variable levels ranging from 1.43 to 211 560.4 pg/mL (mean ± SD; 9158.5 ± 19246.6 pg/mL). Production of Grz B by PBMCs stimulated with Leishmania parasite was not associated with the age or the gender of subjects. Indeed, no correlation was found between subject's age and the Grz B levels (spearman rank correlation r = 0.175; p = 0.002). Likewise, the difference between Grz B levels measured within PBMCs from male subjects and those from female ones was not statistically significant (p = 0.449). Furthermore, no significant difference was found between Grz B levels detected within individuals from El Gtar (OEF; mean ± SD; 6,946 ± 20,295 pg/mL) and those within subjects from Souk Ejjdid (NEF; mean ± SD; 6,269 ± 13,181 pg/mL; p > 0.05). However, in the new endemic focus, Grz B levels were significantly higher within individuals over 6 years old comparing to those within children <6 years old (p < 0.0001; Figure 3A). Moreover, according to the cut-off of positivity of the Grz B levels, the percentage of individuals with high levels of Grz B (35. 8%) was greater in the group over 6 years old than in this under 6 years old (9.3%; Figure 3B). This could reflect the importance of frequency of the contact with the parasite (on time and intensity) on the development of the host's immune response.

We investigated association between Grz B levels, results of LST and the presence of typical ZCL scars. Statistical analysis showed a positive correlation between Grz B levels and the delayed hypersensitivity test (Spearman's rho correlation r = 0.523, p < 0.0001; Figure 4A). However, some discrepancy was noted between these tests. Indeed, we noted that 37 individuals showed variable levels of Grz B despite a negative LST (induration diameter < 5 mm) and conversely 66 individuals showed Grz B levels <2,000 pg/ml although they exhibited positive LST reactivity. As expected, levels of Grz B produced by PBMCs from individuals with a positive LST were higher comparing to those from individuals with negative LST (p < 0.0001; Figure 4B). Similarly, a significant difference was found between Grz B levels within individuals with ZCL scars (Scar⁺) and those without scar (Scar⁻; p < 0.0001; Figure 4C). Moreover, significant differences were found between Grz B levels measured in individuals with a negative LST and no ZCL scar (LST⁻Scar⁻) and those with a positive LST with scars (LST⁺ Scar⁺) or without scars (LST⁺ Scar⁻; p < 0.0001; Figure 4D).

In addition, we found that 138 individuals among the 453 individuals tested showed Grz B levels >2,000 pg /ml. These high levels were specifically found within individuals with positive LST and those with ZCL scars (Figures 4E,4F). As described in Table 3, statistical analysis showed a positive association between levels of Grz B and the positivity of LST (χ² = 112.49, df = 1, p < 0.0001) and the presence of scars (χ² = 49.26, df = 1, p < 0.0001).

To investigate the protective effect of the host's immune status on the development of ZCL, we performed a clinical follow-up of the 453 individuals, between the month of September 2001 and April 2002, to diagnose new lesions of ZCL. We noticed that, after one season of transmission, 90 among the 453 subjects (19.9%) developed new ZCL lesions. Number of lesions ranged from 1 to 9 (Figure 5A) and were mostly localized within limbs or face, with a total area ranging from 12.5 to 5985.6 mm² (mean ± SD = 492.7 ± 784.8 mm²) and a mean duration of progression ranging from 71 to 439 days (mean ± SD = 143.7 ± 62 days).

To refine the analysis, we defined a new variable for the expression of the severity of the disease calculated basing on the total number of lesions, the total surface of lesions (taken at their maximum during the various visits) as well as the average duration of evolution of the lesions. The severity score is determined by the formula: [Average duration of lesions (days)*Total lesion′s area (mm2)]                                                                 /[Number of lesions*1000]

We thus categorized ZCL patients into two groups: (GI) with a lesion's severity score comprised within the interval [0–20] and a second group (GII) with a severity score >20. Among the 90 individuals who developed a new ZCL lesion and for whom information on the spontaneous evolution and size of lesions were available (n = 84), 45 developed a mild form of the disease (GI) while 39 individuals presented intermediate or severe forms of the disease (GII; Figure 5B).

Finally, we evaluated the efficacy of the host's factors in protecting against L. major infection. These factors include the presence of typical ZCL scar, the positive LST reactivity and the induction of high levels of Grz B (≥2000 pg/ml). These factors showed a weak efficacy of protection against the development of ZCL lesion. Indeed, protective efficacy was 29, 15, and 22% for the presence of scar, positive LST reactivity and high Grz B levels with a non-significant p (Table 4).

When ZCL patients were categorized into two groups (GI and GII) according to the lesion's severity score, we showed that none of these parameters was protective against the development of mild ZCL (RR > 1). However, all these parameters were highly protective against the development of intermediate or severe leishmaniasis (Table 4). Accordingly, the incidence of the latter forms of leishmaniasis was significantly lower in individuals with typical ZCL scars (relative risk RR = 0.38, Fisher's exact p = 0.047) and presence of scar showed a protective efficacy of 62% (Table 4). Similarly, positive LST reactivity seems to be efficient in protecting against the development of intermediate or severe disease with a protective efficacy equal to 60% (RR = 0.39, Fisher's exact p = 0.009; Table 4). Strikingly, only 2.9% (4/137) of individuals with Grz B levels >2,000 pg/ml developed intermediate or severe forms of ZCL (Severity > 20) while 13.1% (18/137) developed a mild form of the disease with severity score comprised within the interval [0–20]. Thus high levels of Grz B showed an efficacy of 72.8% (relative risk RR = 0.27, Fisher's exact p = 0.003) to protect against such forms of leishmaniasis. Interestingly, together, the positivity of the LST and level of Grz B >2,000 pg/ml, leads to a better efficacy of protection against development of intermediate or severe leishmaniasis (efficacy is 83.6%). Indeed, only 2% (2/100) of LST⁺ individuals, with Grz B levels >2,000 pg/ml, developed intermediate or severe ZCL (Figure 6).

Conventionally, protection during leishmaniasis is associated with the development of a Th1-type immune response that can be demonstrated in humans by positive LST reactivity and specific in vitro T cell responses associated with IFN-γ production (Liew and O'Donnell, 1993; Sassi et al., 1999; Reithinger et al., 2007). We and others have demonstrated the involvement of cytotoxic T cell (CTL) response as part of the acquired adaptive immune response developed against the parasite Leishmania (da Conceição-Silva et al., 1994; Brodskyn et al., 1997; Marry et al., 1999; Russo et al., 1999; Bousoffara et al., 2004). However, the role of the CTL response during Leishmania infections is still controversial. In humans, clinical studies show the presence of a large number of CD8⁺ T cells in the lesions as well as in blood of ZCL patients during the acute phase, but also during the healing process (Da-Cruz et al., 1994, 2002, 2005; Gaafar et al., 1999; Bottrel et al., 2001). In addition, L. major infection induces Th1 and CD8⁺ T cells in human patients and both responses are associated with disease resolution (Nateghi Rostami et al., 2010). Nevertheless, other studies have shown that cytotoxicity is one of the main mechanisms underlying disease induced by L. braziliensis infection (Faria et al., 2009; Dantas et al., 2013; Novais et al., 2013; Santos et al., 2013; Cardoso et al., 2015; Ferraz et al., 2015, 2017; Novais and Scott, 2015). Herein, we used the release of Grz B by PBMC in response to live L.major promastigotes as marker of cytotoxicity and clearly demonstrated that Grz B is a good marker of immunity to L.major infection, but, most importantly it constitutes a good correlate for protection against intermediate and severe form of CL due to L. major infection. This result is extremely important for vaccine development and evaluation.

In this study, we first showed that in endemic foci of L. major infection subjects having a previous contact with the parasite showed the presence of Leishmania-specific cytotoxicity. Indeed, compared to healthy individuals living outside endemic area for L. major transmission, those living in these areas, including subjects with active or healed ZCL but also those apparently healthy with no ZCL scars and no LST reactivity (Scar⁻ LST⁻) showed high levels of Grz B. Interestingly, even in the absence of LST reactivity, Grz B induction may constitute an indicator of a previous contact with the parasite and may reflect an eventual role in control of Leishmania infection. This data is contradictory with those described by Cordosa and collaborators showing the involvement of CD8⁺T cells producing IFN-g rather than those with cytotoxic activity in the control of Leishmania infection (Cardoso et al., 2015). One explanation could be a difference in the nature of Grz B-producing cells between subjects with history of ZCL and those with subclinical infection. Accordingly, we have previously shown that in healed ZCL subjects (Scar⁺LST⁺), the main source of GrB produced in response to stimulation with Leishmania-antigens is CD4⁺ T cells, including T regulatory cells. These CD4⁺ T cells were different from the Th1 cells producing IFN-γ (Naouar et al., 2014).

Furthermore, the higher peripheral Grz B levels in individuals healed from ZCL compared to those with active disease might be attributed to the homing of Grz B-producing cells to the site of infection in the latter group. Accordingly, we have demonstrated high levels of Grz B mRNA as well as the presence of double positive CD8⁺ Grz B⁺ T cells within active ZCL lesions (Boussoffara et al., submitted). In American cutaneous leishmaniasis (ACL), the percentage of CD8⁺ T cells were higher in lesions compared to blood (Conceição-Silva et al., 1990; Da-Cruz et al., 2005). This, associated with an increase in Leishmania-specific T cells in lesions, have been attributed to the migratory process between these compartments Moreover, a predominance of CD4⁺ and CD8⁺ effector memory T cells (T_EM-CD45RO⁺CCR7⁻) have been shown in ACL lesions. An enrichment of T_EM for both CD4⁺ and CD8⁺ T cells sets were observed comparing to blood (de Oliveira Mendes-Aguiar et al., 2016).

In a next step, the measurement of Grz B levels in culture supernatants of PBMCs stimulated with L. major promastigotes was performed during the prospective study of a cohort of 453 individuals living in L.major transmission area. We, thus, recovered in the beginning of the study (before the season of L. major transmission) the associations between the different parameters indicators of an earlier contact with the Leishmania parasite (presence of typical ZCL scars, LST reactivity and production of Grz B). Our results showed some discrepancies between production of Grz B and scars or LST reactivity. The discrepancy between production of Grz B and the presence or absence of scars might be explained by the fact that lesions of ZCL could disappear without leaving scars and that scars of other pathologies could be taken for ZCL scars. However, the discrepancy between Grz B and LST reactivity seems to be more relevant. Thus, a large fraction of individuals (n = 134) living in the endemic area of leishmaniasis showed a diameter of induration equals to zero but variable levels of Grz B which are sometimes very high. This is consistent of the striking results found during the validation of the Grz B test. Indeed, we were struck by the differences in Grz B levels between the so-called “naïve” (Scar⁻ LST⁻) living in an endemic area of leishmaniasis vs. those living outside the endemic areas. These results are extremely important for the selection of naive populations in endemic areas, especially when including adults in vaccination trials. In fact, the selection of individuals Scar⁻LST⁻ does not exclude an eventual previous contact with the parasite which could be tracked otherwise by the Grz B analysis.

Secondly, we explored the protective role of the cytotoxic effector against the development of the disease. Since ZCL is heterogeneous in terms of disease severity (number and size of lesions, duration of spontaneous healing), we hypothesized that host immune response will not protect against the development of leishmaniasis but rather against the severe forms of it. Our objective was achieved through the monitoring of the cohort included in our prospective study. We analyzed the predictive value of a cytotoxic response (investigated by the Grz B assay) in resistance to L. major infection, as well as the positive LST reactivity, usually used as a protective correlate. Immune response was evaluated at the baseline (before the parasite transmission season) and the development of new ZCL cases was monitored actively during the season of emergence of the disease. A gradation of the disease was also made by calculating a new parameter, severity of the disease, basing on the size of lesions as well as their total duration of evolution.

During the season of emergence of the disease, we diagnosed a total of 90 new cases of ZCL among the 453 individuals tested, with mild, intermediate or high severity score. Interestingly, we noticed a significant number (n = 45) of individuals that develop a simple ZCL characterized by a unique or low number of lesion(s) and a rapid spontaneous healing (severity score <20). Surprisingly, we found that neither the positivity of the LST, nor the presence of ZCL scar, nor the cytotoxic activity (Grz B level ≥2,000 pg/ml), were protective against the development of the disease. This can be explained by the fact that in our study, patients have been actively monitored allowing the diagnosis of mild forms of ZCL, often ignored by the patient himself. However, the positive LST reactivity, the presence of ZCL scar and also the cytotoxic activity were protective against the development of intermediate or severe forms of ZCL. Accordingly, we showed that a Grz B level >2,000 pg/ml has a protective efficacy of 72.8% against the development of intermediate or severe forms of ZCL. Such efficacy was greater than those obtained with the presence of ZCL scar and the positivity of LST (62 and 60%, respectively). Interestingly, together positivity of the LST and a cytotoxic response (Grz B level ≥2,000 pg/ml) confer an excellent protection (>80% efficiency) against intermediate or severe forms of ZCL.

Altogether, our study clearly demonstrated that Grz B production may be an indicator of a previous contact with L.major although the negativity of LST and the absence of scars. Such parameter could be used in vaccination trials for the selection of naive populations living in endemic areas. Moreover, our data show that the preexistence of a parasite-specific cytotoxic immune response may confer protection against the development of intermediate or severe forms of ZCL. These results are of crucial importance for anti-Leishmania vaccine design and more generally for the evaluation of their protective efficacy.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.