Conclusions Our data show that vaccination with alum + LAg and saponin + LAg failed to reduce hepatic parasite burden in BALB/c mice. Moreover, whereas alum + LAg immunization also led to vaccine

failure as evidenced in the splenic compartment, saponin + LAg immunization actually resulted in exacerbation of L. donovani infection in this organ. A high IL-4 response coinciding with enhanced IgG1 correlated with a failure of protection in alum + LAg immunized mice, whereas exacerbation of infection in saponin + LAg immunized mice may involve the unbalanced secretion of IL-4 in conjunction with IL-10. Critically, these results highlight that a limitation to administer LAg through the subcutaneous Pexidartinib route cannot be overcome with the use of the human-compatible adjuvants alum or saponin, tested herein. Moreover, vaccines targeting Leishmania, should aim to generate PLX4032 robust IFN-γ, whilst preventing unfavourable increases

of immunosuppressive cytokines including IL-4 and IL-10. We suggest that further detailed examination of the immunoregulatory responses governing IFN-γ, IL-4 and IL-10 production in immunized mice will greatly focus a priori design considerations necessary to speed production of novel leishmanial vaccines. Methods Animals BALB/c mice were bred in the animal facility of Indian Institute of Chemical Biology Kolkata, India, and were between 4–6 weeks of age at the onset of the experiments. All animal studies were performed according to the Committee for the Purpose of Control and Supervision on Experimental Animals (CPCSEA), Ministry of Environment and Forest, Govt. of India, and approved by the animal ethics committee (147/1999/CPSCEA) of Indian Institute of Chemical Biology. Parasite culture L. donovani strain AG83 (MHOM/IN/1983/AG83) was maintained by serial passage in hamsters and BALB/c mice as described elsewhere [4]. Promastigotes were grown and subcultured at 22°C in Medium 199 (pH 7.4) supplemented with 20%

heat inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin, 25 mM HEPES, 100 μg/ml streptomycin sulphate (all from Sigma-Aldrich, St. acetylcholine Louis, MO, USA). Subcultures were undertaken at an average density of 2 × 106 cells/mL. Preparation of LAg and adjuvants LAg was prepared from L. donovani promastigotes as described previously [4]. Briefly, stationary-phase promastigotes, harvested after the third or fourth passage, were washed three times in cold phosphate-buffered EPZ015938 in vitro saline, pH 7.2 (PBS), pelleted and resuspended at a concentration of 20 mg/mL in cold 5 mM Tris–HCl buffer (pH 7.6). The suspension was centrifuged at 2,310 × g for 10 min to obtain crude ghost membrane pellet, resuspended in Tris–HCl buffer and sonicated for 3 min using an ultrasound probe sonicator (Misonix, Farmingdale, NY, USA).