Cocktail of gp63 and Hsp70 induces protection against Leishmania donovani in BALB ⁄ c mice T. KAUR, 1 R. C. SOBTI 2 & S. KAUR 1 1 Department of Zoology, 2 Department of Biotechnology, Panjab University, Chandigarh, India SUMMARY The 63- kDa antigen of Leishmania donovani is a mem- brane-anchored matrix metalloprotease that has been shown to be involved in the infection process. We have shown that this antigen alone generates a Th1 type of protective response that is partial but when the animals are primed with the antigen along with the Hsp70, the level of protec- tion is raised significantly, which is demonstrated by a con- siderable reduction in parasite load of immunized animals when compared to the infected controls. Delayed-type hyper- sensitivity responses to leishmanin were measured as an index of cell-mediated immune response and were found to be higher in immunized animals when compared to the infected controls, the maximum being in the animals immu- nized with cocktail of both the antigens. Maximum IgG2a and minimum IgG1 levels were observed in this group of ani- mals. These animals also generated maximum levels of IFN- c and IL-2 and minimum levels of IL-4 and IL-10 pointing towards the generation of a protective Th1 response and the suppression of the Th2 type of immune response. Keywords gp63, Hsp70, protective efficacy, Th-1, vaccine, visceral leishmaniasis INTRODUCTION Leishmaniasis, a major and increasing public health prob- lem, is an infectious disease caused by dimorphic obliga- tory intracellular protozoan parasites of Leishmania species (1,2). Clinically, it presents a spectrum of diseases in humans, ranging from cutaneous and mucocutaneous to visceral leishmaniasis, the later being the most fatal. The drugs used for the treatment of kala-azar are effective, but they cause toxicity and come with potential side effects. The emerging drug resistance against the causative parasites is also a major obstacle to their control. With no real promising prospects of good antileishmanials in near future, there is an urgent need to develop new and effec- tive vaccines. It is thus imperative to optimize vaccine tar- gets so that the infection in a susceptible host is controlled. Considerable efforts have been made to develop vaccine- induced specific antiparasitic immune responses. While a substantial progress has been made, no acceptable antile- ishmanial vaccine exists against this infection (3). Leish- IIIf is the only subunit vaccine that has reached human clinical trials (4–7). Leishmune is the only vaccine licensed against the canine disease (8). Gp63, a surface membrane glycoprotein, is present on each promastigote of all species of Leishmania in 500 000 copies. Gp63 has protease activity, and this fact together with its high expression in all species of parasite has made it a favourite candidate for virulence and vaccine studies. A study on cytokine gene expression in healing and non- healing cases of cutaneous leishmaniasis indicates that recombinant gp63 might have the potentiality to induce protective immunity and to stimulate the expression of Th1-related cytokines, which is known as an indicator of protective immunity in leishmaniasis (9). Mice inoculated with a single dose of recombinant BCG species expressing the Leishmania surface proteinase gp63 and challenged with infective Leishmania major or Leishmania mexicana promastigotes exhibited significant protection against these two species of the parasite, as well as against amastigotes of L. mexicana, demonstrating that the immune response recognized the intracellular form of the parasite (10). Gp63 gene delivered orally by a vaccine strain of Salmo- nella typhimurium can preferentially induce the develop- ment of Th-1 subset of CD4 + T cells and protective immunity in the highly susceptible BALB ⁄ c mice (11,12). Recombinant gp63 co-administered with CpG ODN in cationic liposomes induced a protective immune response in BALB ⁄ c mice (13). Correspondence: Sukhbir Kaur, Parasitology Laboratory, Department of Zoology, Panjab University, Chandigarh-160014, India (e-mail: puzoology@yahoo.com). Disclosures: None. Received: 3 May 2010 Accepted for publication: 3 August 2010 Parasite Immunology, 2011, 33, 95–103 DOI: 10.1111/j.1365-3024.2010.01253.x Ó 2011 Blackwell Publishing Ltd 95