Comparative Analysis of Cellular Immune Responses in Treated Leishmania Patients and Hamsters against Recombinant Th1 Stimulatory Proteins of Leishmania donovani.

Our prior studies demonstrated that cellular response of T helper 1 (Th1) type was generated by a soluble antigenic fraction (ranging from 89.9 to 97.1 kDa) of Leishmania donovani promastigote, in treated Leishmania patients as well as hamsters and showed significant prophylactic potential against experimental visceral leishmaniasis (VL). Eighteen Th1 stimulatory proteins were identified through proteomic analysis of this subfraction, out of which 15 were developed as recombinant proteins. In the present work, we have evaluated these 15 recombinant proteins simultaneously for their comparative cellular responses in treated Leishmania patients and hamsters. Six proteins viz. elongation factor-2, enolase, aldolase, triose phosphate isomerase, protein disulfide isomerase, and p45 emerged as most immunogenic as they produced a significant lymphoproliferative response, nitric oxide generation and Th1 cytokine response in PBMCs and lymphocytes of treated Leishmania patients and hamsters respectively. The results suggested that these proteins may be exploited for developing a successful poly-protein and/or poly-epitope vaccine against VL.

Leishmania donovani triose phosphate isomerase: a potential vaccine target against visceral leishmaniasis.

Visceral leishmaniasis (VL) is one of the most important parasitic diseases with approximately 350 million people at risk. Due to the non availability of an ideal drug, development of a safe, effective, and affordable vaccine could be a solution for control and prevention of this disease. In this study, a potential Th1 stimulatory protein- Triose phosphate isomerase (TPI), a glycolytic enzyme, identified through proteomics from a fraction of Leishmania donovani soluble antigen ranging from 89.9-97.1 kDa, was assessed for its potential as a suitable vaccine candidate. The protein- L. donovani TPI (LdTPI) was cloned, expressed and purified which exhibited the homology of 99% with L. infantum TPI. The rLdTPI was further evaluated for its immunogenicity by lymphoproliferative response (LTT), nitric oxide (NO) production and estimation of cytokines in cured Leishmania patients/hamster. It elicited strong LTT response in cured patients as well as NO production in cured hamsters and stimulated remarkable Th1-type cellular responses including IFN-ã and IL-12 with extremely lower level of IL-10 in Leishmania-infected cured/exposed patients PBMCs in vitro. Vaccination with LdTPI-DNA construct protected naive golden hamsters from virulent L. donovani challenge unambiguously (∼90%). The vaccinated hamsters demonstrated a surge in IFN-ã, TNF-á and IL-12 levels but extreme down-regulation of IL-10 and IL-4 along with profound delayed type hypersensitivity and increased levels of Leishmania-specific IgG2 antibody. Thus, the results are suggestive of the protein having the potential of a strong candidate vaccine.

Treatment of Leishmania donovani-infected hamsters with miltefosine: analysis of cytokine mRNA expression by real-time PCR, lymphoproliferation, nitrite production and antibody responses.

OBJECTIVES: Miltefosine, an orally effective antileishmanial drug, works directly on the parasite by impairing membrane synthesis and subsequent apoptosis of the parasite and has also been reported to have macrophage-activating functions that aid parasite killing. We investigated the type of immunological responses generated in miltefosine-treated Leishmania donovani-infected hamsters, which simulate the clinical situation of human kala-azar. METHODS: Twenty-five-day-old infected hamsters, treated with miltefosine at 40 mg/kg for 5 consecutive days, were euthanized on days 30 and 45 post treatment (p.t.) and checked for parasite clearance and for real-time analysis of mRNAs of the Th1/Th2 cytokines interferon-γ (IFN-γ), interleukin-12 (IL-12), tumour necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS), IL-4, IL-10 and transforming growth factor-ß (TGF-ß), nitric oxide (NO) production, the lymphocyte transformation test (LTT) and antibody responses. Responses were compared with the normal and Leishmania-infected groups at the same time points. RESULTS: By day 45 p.t. there was a significant increase in the mRNA expression of iNOS, IFN-γ, IL-12 and TNF-α, whereas there were significant decreases in IL-4, IL-10 and TGF-ß in cured hamsters as compared with their infected counterparts. In vitro stimulation of lymphocytes with concanavalin A and soluble Leishmania donovani antigen showed a maximum LTT response and there was a gradual increase in the NO level (∼7-fold compared with infected counterparts). Anti-Leishmania IgG and IgG1 levels, found to be elevated in the infected group, decreased significantly after treatment but there was a significant increase in IgG2 isotype. CONCLUSIONS: Treatment of Leishmania-infected hamsters with miltefosine reverses the Th2-type response into a strong Th1-type immune response.

Transgenic Leishmania donovani clinical isolates expressing green fluorescent protein constitutively for rapid and reliable ex vivo drug screening.

OBJECTIVES: Several Leishmania strains with episomal expression of green fluorescent protein (GFP) require constant drug pressure for its continuous expression and hence limit its use in ex vivo or in vivo systems. The aim of this study was to alleviate this problem by stably integrating the GFP gene into the parasite genome, so as to use these transfectants for ex vivo and in vivo drug screening. METHODS: The GFP gene was integrated downstream of the 18S ribosomal promoter region of Leishmania donovani. After initial selection, GFP-expressing parasites-both sodium stibogluconate (SAG)-susceptible (2001) and -resistant (2039) isolates-were grown without adding G418. The infectivity of these transfectants to macrophages (J774.1) as well as to hamsters was checked. The ex vivo screening assay was standardized using standard antileishmanial drugs. RESULTS: A constitutive and enhanced expression of GFP in promastigote and amastigote stages was achieved for approximately 12 months without any need for drug pressure. These transfectants were highly infective to macrophage cell lines as well as to hamsters, as observed by fluorescence microscopy and flow cytometry (FACS). GFP-tagged promastigotes as well as intracellular amastigotes were found to be highly susceptible to miltefosine, amphotericin B and pentamidine, in a concentration-dependent manner. SAG was inactive against the GFP-promastigotes, as well as SAG-resistant intracellular amastigotes, correlating well with earlier reports. CONCLUSIONS: The GFP-transfectants were found to be suitable for FACS-based ex vivo screening assays. They were also infective to hamsters up to day 60 post-infection.