Elongation Factor-2, a Th1 Stimulatory Protein of Leishmania donovani, Generates Strong IFN-γ and IL-12 Response in Cured Leishmania-Infected Patients/Hamsters and Protects Hamsters against Leishmania Challenge

Abstract

In visceral leishmaniasis, Th1 types of immune responses correlate with recovery from and resistance to disease, and resolution of infection results in lifelong immunity against the disease. Leishmanial Ags that elicit proliferative and cytokine responses in PBMCs from cured/exposed/Leishmania patients have been characterized through proteomic approaches, and elongation factor-2 is identified as one of the potent immunostimulatory proteins. In this study, we report the cloning and expression of Leishmania donovani elongation factor-2 protein (LelF-2) and its immunogenicity in PBMCs of cured/exposed Leishmania-infected patients and hamsters (Mesocricetus auratus). Leishmania-infected cured/exposed patients and hamsters exhibited significantly higher proliferative responses to recombinant Lelf-2 (rLelF-2) than those with L. donovani-infected hosts. The soluble L. donovani Ag stimulated PBMCs of cured/exposed and Leishmania patients to produce a mixed Thl/Th2-type cytokine profile, whereas rLelF-2 stimulated the production of IFN-γ, IL-12, and TNF-α but not IL-4 or IL-10. Further, rLelF-2 downregulated LPS-induced IL-10 as well as soluble L. donovani Ag-induced IL-4 production by Leishmania patient PBMCs. The immunogenicity of rLelF-2 was also checked in hamsters in which rLelF-2 generates strong IL-12– and IFN-γ–mediated Th1 immune response. This was further supported by a remarkable increase in IgG2 Ab level. We further demonstrated that rLelF-2 was able to provide considerable protection (∼65%) to hamsters against L. donovani challenge. The efficacy was supported by the increased inducible NO synthase mRNA transcript and Th1-type cytokines IFN-γ, IL-12, and TNF-α and downregulation of IL-4, IL-10, and TGF-β. Hence, it is inferred that rLelF-2 elicits a Th1 type of immune response exclusively and confers considerable protection against experimental visceral leishmaniasis.

Characterization and identification of calmodulin and calmodulin binding proteins in hemocyte of the black tiger shrimp (Penaeus monodon)

“…For EF2, it was involved in a production of tumor necrosis factor-α that mediated inflammatory process (Gonzalez-Teran et al, 2013). Also, the EF2 stimulated interferon-γ and interleukin-12 production, promoting pathogen elimination (Kushawaha et al, 2011). Although there was no report on Ca 2+ /CaM involved in EF2 function, this factor contained the CaM binding motif according to our computational prediction.…”

Characterization of the Proliferating Cell Nuclear Antigen of Leishmania donovani Clinical Isolates and Its Association with Antimony Resistance

“…Cultures in the logarithmic phase (at an OD 600 of ϳ0.5 to 0.6) were induced for 3 h with 1.0 mM isopropyl-␤-D-thiogalactopyranoside (IPTG) at 37°C. After induction, 1 ml cells was lysed in 100 l sample buffer (50 mM Tris-HCl [pH 6.8], 10% glycerol, 10% SDS, and 0.05% bromophenol blue, with 10% ␤-mercaptoethanol), and whole-cell lysates (WCL) were analyzed by 12% SDS-PAGE (13). The uninduced control cultures were analyzed in parallel.…”

“…Cloning, expression, and purification of rLdPCNA. L. donovani genomic DNA was isolated from 10 8 promastigotes as described earlier (13). The PCNA gene was amplified using Taq polymerase (TaKaRa) lacking 3= to 5= exonuclease activity through PCR using PCNA-specific primers designed on the basis of the Leishmania major PCNA gene sequence (forward, 5=-CATATGATGCTCGAGGCTCAGGTCCAG-3=, and reverse, 5=-GGATCCCTCCGCATCGTCCACCTT-3=, with NdeI and BamHI restriction sites shown as underlined) in a thermocycler (BioRad) under conditions of 1 cycle of 95°C for 4 min, 30 cycles of 95°C for 1 min, 56°C for 30 s, and 72°C for 1 min, and finally 1 cycle of 72°C for 10 min.…”

Immunoadjuvant Chemotherapy of Visceral Leishmaniasis in Hamsters Using Amphotericin B-Encapsulated Nanoemulsion Template-Based Chitosan Nanocapsules

“…The rationale behind the choice of chitosan biopolymer includes its excellent biocompatibility, biodegradability (12), and "generally regarded as safe" (GRAS) approval (13) and its cellmediated immune-enhancing effects, favoring elevated uptake of the carrier system by macrophages (MP) (14). Stimulation of Th1 and suppression of Th2 immune responses are considered a promising therapeutic strategy for leishmaniasis (15), and chitosan has been reported to stimulate MP to produce various proinflammatory cytokines, including interleukin 1 (IL-1), IL-6, tumor necrosis factor alpha (TNF-␣), nitric oxide (NO), and granulocyte-MP colony-stimulating factor (GM-CSF) (14,16,17). Chitosan also induces immunologic adjuvant effects by active binding to the specific receptors on MP (18)(19)(20).…”