Leishmania donovani serine protease encapsulated in liposome elicits protective immunity in experimental visceral leishmaniasis.

This study is aimed to evaluate the protective effect of L. donovani intracellular serine protease (SP-Ld) in combination with Freund's adjuvant and liposomal formulations against experimental visceral leishmaniasis (VL). The animals were immunized with SP-Ld in combination with adjuvant and evaluated for its immunogenicity and protective efficacy against Leishmania donovani. The infection was initially assessed by microscopic examination. Immunogenicity of SP-Ld was measured by detecting protease specific-IgG, IgG1 and IgG2a levels by ELISA. Cytokines levels were measured by ELISA and Reverse Transcription Polymerase Chain Reaction (RT-PCR). The vaccine efficacy of SP-Ld was also evaluated by measuring antibody response and survival potency in hamster model. SP-Ld vaccinated Balb/c mice resulted significant reduction of parasite burden with increased levels of IgG2a and decreased levels of IgG1. SP-Ld vaccination also induced Th1 type immune response with the rise of IL-12, IFN-γ and TNF-α with decreased levels of IL-10 and TGF-ß. Importantly, liposomal incorporated SP-Ld exerted better protection rather than in combination with Freund's adjuvant. Additionally, liposome encapsulated SP-Ld vaccinated hamsters continued to survive beyond 8 months against virulent L. donovani post challenge. Overall, these findings demonstrated SP-Ld as an effective immunogen which opens a new perspective for the generation of potential vaccine candidate against leishmaniasis.

Inhibition of ABC transporters abolishes antimony resistance in Leishmania Infection.

The emergence of antimony (Sb) resistance has jeopardized the treatment of visceral leishmaniasis in various countries. Previous studies have considered the part played by leishmanial parasites in antimony resistance, but the involvement of host factors in the clinical scenario remained to be investigated. Here we show that unlike infection with Sb-sensitive (Sbs) Leishmania donovani, infection with Sb-resistant (Sb r) L. donovani induces the upregulation of multidrug resistance-associated protein 1 (MRP1) and permeability glycoprotein (P-gp) in host cells, resulting in a nonaccumulation of intracellular Sb following treatment with sodium antimony gluconate (SAG) favoring parasite replication. The inhibition of MRP1 and P-gp with resistance-modifying agents such as lovastatin allows Sb accumulation and parasite killing within macrophages and offers protection in an animal model in which infection with Sb r L. donovani is otherwise lethal. The occurrence of a similar scenario in clinical cases is supported by the findings that unlike monocytes from SAG-sensitive kala-azar (KA) patients, monocytes from SAG-unresponsive KA patients overexpress P-gp and MRP1 and fail to accumulate Sb following in vitro SAG treatment unless pretreated with inhibitors of ABC transporters. Thus, the expression status of MRP1 and P-gp in blood monocytes may be used as a diagnostic marker for Sb resistance and the treatment strategy can be designed accordingly. Our results also indicate that lovastatin, which can inhibit both P-gp and MRP1, might be beneficial for reverting Sb resistance in leishmaniasis as well as drug resistance in other clinical situations, including cancer.

Sodium antimony gluconate induces generation of reactive oxygen species and nitric oxide via phosphoinositide 3-kinase and mitogen-activated protein kinase activation in Leishmania donovani-infected macrophages.

Pentavalent antimony complexes, such as sodium stibogluconate and sodium antimony gluconate (SAG), are still the first choice for chemotherapy against various forms of leishmaniasis, including visceral leishmaniasis, or kala-azar. Although the requirement of a somewhat functional immune system for the antileishmanial action of antimony was reported previously, the cellular and molecular mechanism of action of SAG was not clear. Herein, we show that SAG induces extracellular signal-regulated kinase 1 (ERK-1) and ERK-2 phosphorylation through phosphoinositide 3-kinase (PI3K), protein kinase C, and Ras activation and p38 mitogen-activated protein kinase (MAPK) phosphorylation through PI3K and Akt activation. ERK-1 and ERK-2 activation results in an increase in the production of reactive oxygen species (ROS) 3 to 6 h after SAG treatment, while p38 MAPK activation and subsequent tumor necrosis factor alpha release result in the production of nitric oxide (NO) 24 h after SAG treatment. Thus, this study has provided the first evidence that SAG treatment induces activation of some important components of the intracellular signaling pathway, which results in an early wave of ROS-dependent parasite killing and a stronger late wave of NO-dependent parasite killing. This opens up the possibility of this metalloid chelate being used in the treatment of various diseases either alone or in combination with other drugs and vaccines.