Immunogenicity and efficacy of recombinant 78 kDa antigen of Leishmania donovani formulated in various adjuvants against murine visceral leishmaniasis

Objective: To analyze the protective efficacy of recombinant 78 kDa antigen of Leishmania donovani in combination with two adjuvants, that is, cationic liposomes or MPL-A against visceral leishmaniasis in BALB/c mice. Methods: The genomic DNA of promastigotes was isolated and 583 bp of T cell epitopes of gene encoding 78 k Da was amplified using specific primers. The amplified gene was cloned into p ET28 c, transformed into Escherichia coli BL21(DE3) and got expressed after IPTG induction. The recombinant protein was then purified using Ni-NTA and named r78. Three groups of mice were immunized with 10 μg of r78 plus MPL-A, r78 encapsulated in positively charged liposomes and control animals immunized with PBS. Two booster doses were given with the respective vaccine at an interval of 2 weeks each. Mice were challenged with 1×107 Leishmania promastigotes and sacrificed on different post infection/challenge days. Results: Immunization with r78 along with MPL-A and liposomeencapsulated r78 brought a significant reduction in parasite load. In comparison to the infected controls, the parasite load declined by 96.2% in mice immunized with r78 plus MPL-A and 97.23% in animals immunized with liposome-encapsulated r78. The immunized animals also exhibited profound DTH response. The serum antibody responses increased from 15 to 90 days post infection/challenge. Immunized animals showed greater IgG2 a levels and lesser Ig G1 levels in comparison to the infected controls. The splenocytes from immunized mice were cultured, stimulated with r78 and analyzed for cytokine profile. The levels of IL-2 and IFN-γ were greater in immunized animals as compared to control mice. Conclusions: The study proves that r78 in combination with suitable adjuvants is a potential vaccine candidate and may be instrumental in control of visceral leishmaniasis.

Immunostimulatory role of rBmHSP60 from filarial parasite Brugia malayi

Objective:To evaluate the immunostimulatory potential of crossreactive molecule heat shock protein 60(HSP60)of filarial parasite Brugia malayi and Leishmania donovani.Methods:HSP60 of Brugia malayi(BmHSP60)was amplified using gene-specific primer,cloned in p Tri Ex4 vector,expressed in BL21-DE3 cells,and recombinant HSP60(rHSP60)of~65 k Da was purified by affinity chromatography using Ni-NTA column.The recombinant protein was desalted by the dialysis membrane,and the presence of endotoxin level was determined by Limulus amebocyte lysate assay.The recombinant protein was tested for cell proliferation,nitric oxide release,expression of Th1 and Th2 cytokines,and transcription factors(STATs)in vitro using murine macrophage cell line(J774 A.1).Results:Higher cell proliferation indicated that BmHSP60 had immunostimulatory potential.rBmHSP60 exposure upregulated the expression of iNOS,STAT1,STAT4,Th1 cytokines(IFN-γ,TNF-α,IL-12),and nitric oxide release.In addition,no remarkable change was observed in the expression of IL-6,IL-10,and STAT3 in macrophage cell line J774 A.1.The ELISA analysis showed the levels of IFN-γ,TNF-α,and IL-12 were upregulated while IL-10 level was downregulated,revealing that BmHSP60 triggered a Th1 immune response.Conclusions:Our study demonstrates that rBmHSP60 has immunogenic properties which effectively enhances the Th1 type immune responses,and can be used as an immunoprophylactic agent against leishmaniasis.Furthermore,in vivo studies are in progress to determine the protective role of rBmHSP60 against Leishmania donovani infection in a mouse model.

Immunogenicity and protective efficacy of DNA vaccine against visceral leishmaniasis in BALB/c mice

The current study was designed to examine the protective efficacy of DNA vaccines based on gp63 and Hsp70 against murine visceral leishmaniasis. Inbred BALB/c mice were immunized subcutaneously twice at an interval of three weeks with pcDNA3.1 （＋） encoding T cell epitopes of gp63 and Hsp70 individually and in combination. Animals were challenged intracardially with 107 promastigotes ofLeishmania donovani 10 days post immunization and sacrificed 1,2 and 3 months post challenge. The immunized animals revealed a significant reduction （P 〈 0.05） in splenic and hepatic parasite burden as compared to the infected controls. Maximum reduction in parasite load （P 〈 0.05） was observed in animals treated with a combination ofpcDNA/gp63 and pcDNA/Hsp70. These animals also showed heightened DTH response, increased IgG2a, elevated Thl cytokines （IFN-γ and IL-2） and reduced IgG 1 and IL-10 levels. Thus, mice immunized with the cocktail vaccine exhibited significantly greater protection in comparison to those immunized with individual antigens.