IL-4 have also been described for L. donovani resistance [18]. Moreover, recent studies in CL indicate that induction of high levels of the regulatory cytokine IL-10 can cause vaccine failure even in the presence of high levels of IFN-c [19]. Thus, one of the biggest challenges for development of a vaccine against VL is to unravel the prechallenge predictor for vaccine success. Our prior observations demonstrated that antigens of L. donovani promastigotes (LAg) in cationic liposomes could significantly reduce parasite load in BALB/c mice and hamsters after challenge infection [20]. We also found that LAg had strong serologic reactivity with Indian VL patient sera. While dissecting the humoral immune responses to the dominant components of LAg by Western blot analysis, we found that polypeptides with molecular masses of 31, 34, 51, 63, 72 and 91-kDa were reactive with all the kala-azar patients’ sera prior to treatment. Although after successful treatment with sodium antimony gluconate (SAG) decreases in the frequency of recognition and the intensities of bands were observed, the bands were still reactive with posttreatment sera with high frequencies ranging from 73% to 100% [21]. These observations provide an insight into the vaccine potential of these antigens. Our previous report demonstrated that 63-kDa antigen entrapped in cationic liposomes induced reduction in parasite burden in BALB/c mice [22], and liposomal 34-kDa antigen was found to be ineffective to reduce parasite level in the same murine model (unpublished observation). Thus, in the present study we purified and identified the remaining four polypeptides of approximately 91, 72, 51 and 31-kDa and compared the challenge outcome with L. donovani and immune responses in BALB/c mice to identify key immunological correlates of protection in VL. Methods Animals and parasites BALB/c mice, bred in the Institute animal facility (Kolkata, India), were 4–6 weeks old at the onset of the experiments. The studies were approved by IICB Animal Ethical Committee and mice were handled according to their guidelines. L. donovani strain AG83 (MHOM/IN/1983/AG83) promastigotes were grown at 22uC in Medium 199 supplemented with penicillin G sodium (l00 U/ml), streptomycin sulfate (100 mg/ml) and 10% heat inactivated fetal bovine serum (FBS) (Sigma-Aldrich, St. Louis, MO) and subcultured in the same medium at an average density of 26106 cells/ml [20]. Preparation of antigens Leishmanial antigens (LAg) were prepared from L. donovani promastigotes as described earlier [20]. Briefly, stationary-phase promastigotes, harvested after the third or fourth passage, were washed three times in cold 20 mM phosphate-buffered saline (PBS), pH 7.2, and resuspended at a concentration of 1.0 g of cell pellet in 50 ml of cold 5 mM Tris-HCl buffer, pH 7.6. The suspension was vortexed and centrifuged at 2,3106g for 10 min. The crude ghost membrane pellet thus obtained was resuspended in the same Tris buffer and sonicated for 3 min by ultrasound probe sonicator (Misonix, Farmingdale, NY). The suspension was centrifuged at 5,1906g for 30 min, and the supernatant containing the leishmanial antigens was harvested and stored at 270uC until use. The amount of protein obtained from a 1.0-g cell pellet, as assayed by the method of Lowry et al. [23], was approximately 10 mg. SDS-PAGE and electroelution The LAg was subjected to sodium dodecyl sulfate–10% polyacrylamide gel electrophoresis (SDS–10% PAGE) by the Novel Antigens of L. donovani method of Laemmli et al., 1970 [24] and stained with Coomassie blue. The proteins with molecular mass of 91, 72, 51 and 31-kDa were eluted by electrophoresis in running buffer (0.025 M Tris, 0.192 M glycine, 1% SDS) using a Electro-Eluter (model 422; Bio- Rad) at 10 mA for 5 h. After elution, the proteins were dialyzed, lyophilized and resuspended in PBS. Proteins were further visualized by SDS-PAGE and silver staining [25]. The proteins were quantified by Lowry’s method [23]. Entrapment of antigens in liposomes Cationic liposomes were prepared with distearoyl phosphati- dylcholine (DSPC) (27 mmole), cholesterol (Sigma-Aldrich) and stearylamine (Fluka, Buchs, Switzerland) at a molar ratio of 7:2:2 as described previously [22]. Empty and antigen entrapped liposomes were prepared by the dispersion of lipid film in either 1 ml PBS alone or containing 200 mg/ml antigen respectively. The mixture was vortexed and the suspension sonicated for 30 sec by an ultrasound probe sonicator (Misonix). Liposomes with entrapped antigens were separated from excess free antigen by three successive washing in PBS with ultracentrifugation (105,0006g, 60 min, 4uC) [25]. The encapsulation efficiency was determined by the method of Lowry et al., in the presence of 10% SDS [23]. The level of encapsulation ranged between 50 to 60%. Immunization of mice and challenge infection The experimental groups consisted of 4–6 weeks old BALB/c mice. Mice were immunized by intraperitoneal injections of 2.5 mg purified proteins in PBS or incorporated in liposome in a total volume of 200 ml. Animals receiving PBS or empty liposomes served as controls. Mice were boosted two times at 2-week intervals. Ten days after the last booster, serum samples were collected, and spleens were removed aseptically for the analysis of humoral and cellular responses after immunization. Ten days after the final immunization rest of the mice were challenged with 2.56107 freshly transformed stationary-phase promastigotes in 200 ml PBS injected intravenously via the tail vein as described earlier [20,22]. After 3 months of challenge infection, the mice were sacrificed to determine the parasite load in liver and spleen [22]. The course of infection was monitored by the microscopic examination of Giemsa-stained impression smears of liver and spleen. The parasite load was expressed as Leishman-Donovan units and was calculated by the following formula: number of amastigotes per 1000 cell nuclei6organ weight (mg) [26]. Assessment of delayed type hypersensitivity response (DTH) Delayed type hypersensitivity (DTH) was determined as an index of cell mediated immunity. The response was evaluated by measuring the difference in the footpad swelling at 24 h following intradermal inoculation of the test footpad with 50 ml of LAg (800 mg/ml) and the swelling of the control (PBS injected) footpad with a constant pressure caliper (Starrett Company, Athol, MA) [20]. Cell proliferation and cytokine assays The spleens were aseptically removed from the immunized and infected BALB/c mice and single cell suspensions were prepared in RPMI 1640 supplemented with 10% FBS, l00 U/ml penicillin G sodium, 100 mg/ml streptomycin sulfate and 50 mM b- mercaptoethanol (Sigma-Aldrich) (complete medium). RBCs were removed by lysis with 0.14 M Tris buffered NH4Cl. The remaining cells were washed twice with culture medium and viable mononuclear cell number was determined by counting Trypan blue unstained cells in a hemocytometer. Then the cells PLoS ONE | www.plosone.org 2 June 2009 | Volume 4 | Issue 6 | e5820