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. 2018 Nov 13:8:397.
doi: 10.3389/fcimb.2018.00397. eCollection 2018.

Immunity Against Leishmania major Infection: Parasite-Specific Granzyme B Induction as a Correlate of Protection

Thouraya Boussoffara  1   2 , Sadok Chelif  1   2 , Melika Ben Ahmed  1   2 , Mourad Mokni  3 , Afif Ben Salah  1   2 , Koussay Dellagi  1   2 , Hechmi Louzir  1   2
Affiliations

Immunity Against Leishmania major Infection: Parasite-Specific Granzyme B Induction as a Correlate of Protection

Thouraya Boussoffara et al. Front Cell Infect Microbiol. .

Abstract

Zoonotic cutaneous leishmaniasis (ZCL) caused by Leishmania (L.) major infection is characterized by different clinical presentations which depend in part on the host factors. In attempt to investigate the impact of the host's immune response in the outcome of the disease, we conducted a prospective study of 453 individuals living in endemic foci of L. major transmission in Central Tunisia. Several factors were assessed at the baseline including (i) the presence of typical scars of ZCL, (ii) in vivo hypersensitivity reaction to leishmanin, and (iii) the in vitro release of granzyme B (Grz B) by peripheral blood mononuclear cells (PBMC) in response to stimulation with live L. major promastigotes. After one season of parasite's transmission, repeated clinical examinations allowed us to diagnose the new emerging ZCL cases. Heterogeneity was observed in terms of number of lesions developed by each individual as well as their size and spontaneous outcome, which led us to establish the parameter "severity of the disease." The efficacy of the presence of typical ZCL scar, the leishmanin skin test (LST) positive reactivity and the high levels of Grz B (≥2 ng/ml), in the protection against the development of ZCL were 29, 15, and 22%, respectively. However, these factors were more efficient against development of intermediate or severe forms of ZCL. Levels of Grz B >2 ng/ml showed the best efficacy of protection (equals to 72.8%) against development of these forms of ZCL. The association of such parameter with the positivity of the LST exhibited a better efficacy (equals to 83.6%). In conclusion, our results support the involvement of Leishmania-specific cytotoxic cellular immune response in host protection against Leishmania-infection. This factor could be of great interest in monitoring the success of vaccination against human leishmaniasis.

Keywords: Leishmania major; correlate of protection; granzyme B; re-infection; specific-cytotoxicity.

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Figures

Figure 1
Figure 1
Timeline showing progress of the prospective study. Four hundred and fifty three participants living in endemic foci of zoonotic cutaneous leishmaniasis (ZCL) in Central and Southwestern Tunisia (Gafsa and Sidi Bouzid) were followed up throughout one season of L. major transmission. Several parameters such as leishmanin skin test (LST) or the presence of typical ZCL scar were monitored at the beginning of the study. Peripheral blood samples were obtained from each donor and will be used to analyze host cytotoxic immune response. Repeated clinical examinations were done to diagnose the new cases of ZCL emerging after one transmission season (September 2001–April 2002).
Figure 2
Figure 2
Granzyme B levels induced in response to stimulation with Leishmania major. (A) Grz B is measured in culture supernatants of PBMCs incubated for 5 days with live promastigotes L. major at a ratio of 3 promastigotes per cell. We used PBMCs from patients with active ZCL, healed ZCL subjects or from healthy individuals living in endemic focus for transmission of L. major or outside these areas. Horizontal bars represent median values. (B) Histograms represent the number of subjects categorized in Grz B+ and Grz B−, basing on the cut-off fixed at 2,000 pg/mL. (C) Association between Grz B and IFN-γ levels measured in the same supernatants (D,E) Association between Grz B levels and results of leishmanin skin test (LST), expressed as diameter of induration and the lymphoproliferative response to soluble Leishmania antigens (SLA), expressed as Δcpm. Horizontal dashed lines indicate cut-off for Grz B level, LST and proliferation fixed at 2,000 pg/ml, 5 mm and 5,000 cpm, respectively.
Figure 3
Figure 3
Evaluation of granzyme B levels for subjects included in the prospective study. (A) Grz B levels measured in culture supernatants of PBMCs from individuals, categorized basing on their home focus and their age. Horizontal bars represent median values. (B) Percentage of subjects showing high levels of GrzB (≥2,000 pg/mL; Grz B+) and those with levels <2,000 pg/mL (Grz B−). OEF: old endemic focus; NEF, New endemic focus.
Figure 4
Figure 4
Association between granzyme B levels, LST reactivity and presence of typical ZCL scar. (A) Grz B level expressed in function of result of LST. n number of individual for each group. (B–D) Histograms represent mean values of Grz B levels measured in culture supernatants of PBMCs from individuals, categorized basing on categorical results of LST (LST+/LST−) and presence or absence of ZCL scars (scar+/scar−). (E,F) Categorial result for Grz B expression within individuals categorized basing on LST results (LST+/LST−) or presence or absence of scar (scar+/scar−). Grz B+: Grz B levels ≥2 ng/ml, Grz B−: Grz B levels <2 ng/ml. LST+, Diameter of induration ≥5 mm; LST−, Diameter of induration <5 mm.
Figure 5
Figure 5
Prevalence of ZCL after one transmission season of L. major. Prevalence of the ZCL according to (A) the number of lesions developed or (B) the severity of the disease. G0, no ZCL lesion; GI, individuals developing a mild ZCL (severity score contained with the interval [0–20]); GII, individuals developing an intermediate or severe leishmaniasis (severity score ≥20).
Figure 6
Figure 6
Impact of the host's immune response on the prevalence of ZCL. Expression of severity score in function of result of LST reactivity, expressed as diameter of induration (mm) and those of Grz B induction expressed in pg/mL.
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