were cultured in triplicate in a 96 well flat bottom plate (Nunc, Roskilde, Denmark) at a density of 26105 cells/well in a final volume of 200 ml complete medium and stimulated with antigens (2.5 mg/ml). The cells were incubated for 96 h at 37uC in a humified chamber containing 5% CO2. Cells were pulsed with 1 mCi of [3H ]-Thymidine (Amersham Biosciences, Buckingham- shire, UK) per well 18 h before they were harvested on glass fiber paper. Thymidine uptake was measured in a b-scintillation counter (Beckman Instruments, Fullerton, CA) [25]. After 72 h incubation, culture supernatants were collected and the concen- tration of IFN-c, IL-4, IL-12p40 and IL-10 (BD Pharmingen, San Diego, CA) were quantitated by ELISA in accordance with the manufacture’s instructions [27]. Determination of antibody response Sera from immunized and infected animals were analyzed by ELISA for the presence of antigen specific antibodies. In brief, 96- well microtiter plates (Maxisorp, Nunc) were coated with antigens (10 mg/ml) diluted in 20 mM phosphate buffer (pH 7.5) overnight at 4uC. The plates were blocked with 1% BSA in PBS at room temperature for 3 h to prevent nonspecific binding. After washing with PBS containing 0.05% Tween 20 (Sigma-Aldrich) (PBST) the plates were incubated overnight with 1:1000 dilution of mice sera at 4uC. Next day, the plates were again washed with PBST and incubated further for 3 h at room temperature with horseradish peroxidase conjugated goat antimouse IgG1 and IgG2a (BD Pharmingen) diluted 1:1000 in blocking buffer. The plates were washed and substrate solution (o-phenylene diamine dihydrochlo- ride, 0.8 mg/ml in 0.05 M phosphate-citrate buffer, pH 5.0, containing 0.04% H2O2 ) (100 ml) was added for 30 min and the absorbance was read in an ELISA plate reader (Thermo, Waltham, MA) at 450 nm [20]. Mass spectrometry Protein samples were separated by SDS-PAGE and visualized with Coomassie blue or silver staining (ProteoSilverTM Plus silver stain kit, Sigma-Aldrich). The in-gel digestion of proteins was carried out according to the manufacturer’s manual (Pierce, Rockford, IL). The desired band was excised and destained. The possible disulfide bonds were reduced with tris (2-carboxyethyl) phosphine (TCEP) and alkylated with iodoacetamide. Then the gel pieces were dehydrated with acetonitrile and rehydrated with 100 ng trypsin (Promega, Madison, WI) in 25 mM ammonium bicarbonate solution and were incubated at 37uC overnight. The tryptic fragments were extracted from the gel by adding 1% trifluroacetic acid. Then, the peptides were purified with C18 reversed-phase minicolumn filled in a micropipette tip, ZipTip C18 (Millipore, Bedford, MA). Purified peptides (0.5 ml) were cocrystallized with a-cyano-4-hydroxy cinnamic acid matrix (0.5 ml) (Applied Biosystems, Foster City, CA) on a matrix-assisted laser desorption ionization (MALDI) target plate. Both mass spectrometry (MS) and MS/MS spectra were acquired by matrix- assisted laser desorption ionization–time of flight (MALDI-TOF/ TOF) Mass Spectrometer (Applied Biosystems 4800 Proteomics Analyzer). All spectra were collected in the reflector mode. Calibration was updated before each acquisition using a standard peptide mixture according to instrument protocol. Database searching for protein identification was performed with mass spectrometry data using GPS Explorer (Applied Biosystems) software with MASCOT (Matrix Science) search engine. N-terminal sequencing The antigens were subjected to SDS-PAGE electrophoresis, and electrotransferred onto a polyvinylidene difluoride membrane Novel Antigens of L. donovani (Immobilon P; Millipore) in 10 mM 3-[cyclohexylamino]-1- propanesulfonic acid and 10% methanol, pH 11.0. The bands on the membrane were excised after staining according to manufacturer’s instructions. The N-terminal amino acid sequence was determined by Edman degradation chemistry using a Procise 492 Protein sequencer (Applied Biosystems) [22]. Statistical analysis One-way analysis of variance (ANOVA) and Tukey’s multiple comparisons post-test were used for the analysis of parasite burden and antibody response data using Graph Pad Prism version 4.0 (Graphpad Software, v. 4.0, San Diego, CA). The statistical analyses for other experiments before and after infection were made by a two-way ANOVA. Corrections to the level of significance were done using Bonferroni’s method. Results with p,0.05 were considered to be statistically significant. For correlation analysis, all datasets were calculated for correlation efficiency and were considered significant at p,0.05. Spearman’s rank correlation coefficient values were evaluated and represented (r). Results Parasite burdens in vaccinated BALB/c mice after L. donovani challenge infection Proteins from LAg ranging from 91 to 31-kDa were purified by eletroelution from 10% SDS-PAGE gel and then visualized on SDS-PAGE followed with silver staining (Figure 1). BALB/c mice were immunized intraperitoneally with antigens LD91 (91-kDa), LD72 (72-kDa), LD51 (51-kDa) and LD31 (31-kDa) entrapped in cationic liposomes as the adjuvant. The vaccination was repeated twice at 2-week intervals and the mice were challenged intravenously with L. donovani promastigotes 10 days after the last immunization. Control mice were injected with the empty liposomes or PBS. We and others have found that infection with the Indian strain, L. donovani AG83 in BALB/c mice results in a progressive infection in the liver and spleen, corresponding with hepato and splenomegaly [22,27–30]. Mice were therefore sacrificed after 3 months when parasite loads were well expressed in both liver and spleen. In liver, the greatest degree of reduction in parasite burden was observed in mice immunized with liposomal LD31 (74%) followed by LD51 (72%) (Figure 2A). Although immunization with liposomal LD31 induced higher levels of reduction than liposomal LD51, the difference was not statistically significant. Vaccination with liposomal LD72 (65%) conferred reduction in parasite load to a moderate level. Immunization with LD91 in liposomes (46%) also reduced parasite load significantly in mice but to the lowest degree (p,0.001 compared to controls). In BALB/c mice persistence of L. donovani in the spleen causes concomitant development of considerable organ-specific pathology similar to that seen in the human kala- azar. It was, therefore, important to evaluate the effect of vaccination in this organ. Similar to liver, mice immunized with liposomal LD31 (77%) and LD51 (75%) demonstrated highest reduction in splenic parasite burden with a moderate level in mice immunized with LD72 (67%) and lowest but significant level with LD91 (49%, p,0.001 compared to controls) (Figure 2B). It may be noted that the reduction in parasite burden by the different liposomal antigens were maintained in both the organs. DTH and splenocyte proliferative responses in vaccinated mice before and after challenge infection DTH, an index of cell- mediated immunity in vivo, and antigen induced proliferation of splenocytes in vitro were evaluated in different vaccinated mice 10 days after the last immunization. Among the different vaccinated groups, liposomal LD31 and LD51 immunized groups exhibited PLoS ONE | www.plosone.org 3 June 2009 | Volume 4 | Issue 6 | e5820