The crude ghost membrane pellet
thus obtained was resuspended in the same Tris buffer and sonicated three times for 1 min each at 4°C in an ultrasonicator (Misonix, New York, USA). The suspension was finally centrifuged for 30 min at 5,190 × g, and the supernatant containing leishmanial antigens (LAg) was harvested and stored at -70°C until used. The amount of protein obtained selleck products from a 1.0 g cell pellet was approximately 14 mg, as assayed by the method of Lowry et al.  with bovine serum albumin as the standard, in the presence of 1% sodium dodecyl sulphate and appropriate blanks. Adjuvants Positively charged liposomes were prepared with egg lecithin, cholesterol, and stearylamine (7:2:2 molar ratio), respectively as reported earlier . MPL (0.5 mg) plus trehalose dicorynomycolate Selleck 3Methyladenine (TDM) (0.5 mg) in 2% oil (squalene)-Tween 80-water was purchased from Sigma-Aldrich Corp., St. Louis, USA.
Briefly, each vial was reconstituted with 1 ml saline and mixed at 1:1 ratio with LAg in PBS and administered in mice as 50 μg/dose. The mean particle size of the emulsion droplets was 128 ± 6.65 as determined by Zetasizer Nano-ZS (Malvern Instruments, Worcestershire, UK). Bacillus Calmette Guerin (BCG) (Pasteur Institute, Paris, France) was diluted in PBS mixed at 1:1 ratio with LAg in PBS prior to injection to an administrable dose of 5 × 104 cells/mice. Entrapment of leishmanial antigens into cationic liposomes For encapsulation of the LAg in the liposomal vesicles the lipid film was dispersed
in PBS containing 1 mg/ml LAg and sonicated for 30 s in an ultrasonicator (Misonix) . Liposomes with entrapped LAg were separated from excess free materials by three successive washing in PBS with ultracentrifugation (105,000 × g, 60 min, 4°C). The mean size of the LAg entrapped liposomes was 337.3 ± 10.2 as determined by Zetasizer Nano-ZS (Malvern Instruments). The presence of antigen could Pregnenolone not influenced the size of the vesicles (empty vesicles mean size 306.8 ± 2.6). The protein content entrapped into liposomes was estimated by the method described by Lowry et al. . The phospholipid content of liposomes was 15.5 mg/ml as determined using the Stewart assay . The average amount of LAg associated per mg of egg lecithin was 33 μg. Vaccination and challenge infection BALB/c mice were vaccinated by three intraperitoneal injections of 20 μg of free LAg, incorporated in liposomes, or associated with other adjuvants at 2-week intervals in a total volume of 200 μl. PBS and only Staurosporine clinical trial adjuvant treated animals were included as controls. Ten days after last immunization the animals were challenged intravenously with 2 × 107 freshly transformed promastigotes . Evaluation of infection At the times designated in Results, the course of infection was evaluated by microscopic examination of Giemsa-stainted impression smears of liver and spleen samples.