Phlebotomus papatasi Yellow-Related and Apyrase Salivary Proteins Are Candidates for Vaccination against Human Cutaneous Leishmaniasis  Aymen Tlili, Soumaya Marzouki, Emna Chabaane, Maha Abdeladhim, Wafa Kammoun-Rebai, Rahma Sakkouhi, Nabil Belhadj Hmida, Fabiano Oliveira, Shaden Kamhawi, Hechmi Louzir, Jesus G. Valenzuela, Mélika Ben Ahmed  Journal of Investigative Dermatology  Volume 138, Issue 3, Pages 598-606 (March 2018) DOI: 10.1016/j.jid.2017.09.043 Copyright © 2017 The Authors Terms and Conditions

Figure 1 Western blot analysis of the fractionated salivary preparations. Salivary gland extract (SGE) and salivary fractions (<30 and >30 kDa) were run on a 15% SDS-PAGE gel. SGE and fractionated salivary extracts were incubated with positive anti-SGE human sera from donors living in an area of high prevalence of P. papatasi. Western blot analysis was performed using an anti-human IgG antibody. One representative experiment is shown. Journal of Investigative Dermatology 2018 138, 598-606DOI: (10.1016/j.jid.2017.09.043) Copyright © 2017 The Authors Terms and Conditions

Figure 2 Cellular immune response against the fractionated salivary preparations. PBMCs were from eight volunteer donors living in endemic areas for zoonotic cutaneous leishmaniasis in Tunisia and selected according to their positive PBMC immune response to total SGE stimulation. CD4 (PBMCs depleted of CD8 T cells) and CD8 (PBMCs depleted of CD4 T cells) T cells were stimulated for 5 days with SGE and fractionated salivary extract (>30 and >30 kDa) in 96-well plates. (a) Proliferative response was assessed by (3H) thymidine uptake after 5 days of culture. Results are expressed as an index of proliferation (mean counts of triplicates in antigen-stimulated cultures/mean counts of triplicates in unstimulated cultures) in all tested individuals. (b) IFN-γ secretion was evaluated in the supernatants of cultures by an ELISA test at day 3. Results were expressed as a ratio of cytokine levels in stimulated to unstimulated cultures. PBMC, peripheral blood mononuclear cell; SGE, salivary gland extract. Journal of Investigative Dermatology 2018 138, 598-606DOI: (10.1016/j.jid.2017.09.043) Copyright © 2017 The Authors Terms and Conditions

Figure 3 Proliferative response and IFN-γ production of PBMCs transfected with different plasmids of interest. PBMCs of 10 donors living in endemic areas for zoonotic cutaneous leishmaniasis in Tunisia and selected according to their positive PBMC response (proliferation and IFN-γ production) to total salivary gland extract stimulation were transfected with plasmids encoding PpSP15, PpSP28, PpSP34, PpSP36, PpSP42, and PpSP44. (a) Nucleofected cells were cultured in 96-well plates (105/well) and the uptake of (3H) thymidine was measured at day 5. Results were expressed as a proliferation index: mean counts of triplicates in cells nucleofected with plasmids/mean counts of triplicates in nucleofected without plasmids in 7 of 10 tested individuals. (b) Nucleofected cells were cultured in 24-well plates. The supernatants of transfection were collected at 72 hours, and IFN-γ secretion was evaluated by an ELISA test. IFN-γ secretion was detected in 7 10 donors, and results were expressed as IFN-γ concentration (pg/ml). PBMC, peripheral blood mononuclear cell. Journal of Investigative Dermatology 2018 138, 598-606DOI: (10.1016/j.jid.2017.09.043) Copyright © 2017 The Authors Terms and Conditions

Figure 4 Cytokine multiplex analysis of PBMC-transected supernatants. Seven cytokines (IFN-γ, IL-17, IL-10, IL-4, IL-6, tumor necrosis factor-α, and IL-2) were monitored using a Th1/Th2/Th17 7-Plex assay in the supernatants of PBMC-transected cell cultures of five donors exhibiting a cellular immune response with PPTSP36, PPTSP42, and PPTSP44. Results were expressed as the mean of the cytokine concentration obtained in all tested donors. PBMC, peripheral blood mononuclear cell; Th1, T helper type 1. Journal of Investigative Dermatology 2018 138, 598-606DOI: (10.1016/j.jid.2017.09.043) Copyright © 2017 The Authors Terms and Conditions

Figure 5 Proliferative response and IFN-γ production of PBMCs stimulated with the recombinant form of PPTSP44. Freshly purified PBMCs were isolated from 10 volunteer donors living in endemic areas for zoonotic cutaneous leishmaniasis in Tunisia and selected according to the positive cellular immune response of their PBMCs to total salivary gland extract stimulation. Five healthy volunteers with no history of leishmaniasis or traveling to sand fly endemic areas have also been tested. PBMCs were stimulated in 96-well plates with the recombinant protein PPTSP44 at 2 μg/ml during 5 days. (a) Proliferative response was assessed by (3H) thymidine uptake. Results were expressed as a proliferation index (mean counts of triplicates in antigen-stimulated cultures/mean counts of triplicates in unstimulated cultures). (b) The supernatants of stimulated PBMCs were collected at 72 hours and IFN-γ secretion was evaluated by an ELISA test. PBMC, peripheral blood mononuclear cell. Journal of Investigative Dermatology 2018 138, 598-606DOI: (10.1016/j.jid.2017.09.043) Copyright © 2017 The Authors Terms and Conditions