Immunobiology 218 (2013) 114–126 Contents lists available at SciVerse ScienceDirect Immunobiology jo u rn al homepage: www.elsevier.de/imbio 115 kDa serine protease confers sustained protection to visceral leishmaniasis caused by Leishmania donovani via IFN- induced down-regulation of TNF- mediated MMP-9 activity Rajdeep Choudhury a,1 , Partha Das a , Tripti De b , Tapati Chakraborti a,∗ a Department of Biochemistry and Biophysics, University of Kalyani, Kalyani 741235, West Bengal, India b Infectious Disease and Immunology Division, Indian Institute of Chemical Biology, Kolkata 700032, India a r t i c l e i n f o Article history: Received 1 September 2011 Received in revised form 8 February 2012 Accepted 8 February 2012 Keywords: Extracellular serine protease Immunization Leishmania donovani Matrix metalloprotease-9 Visceral leishmaniasis a b s t r a c t Visceral leishmaniasis caused by the intracellular parasite Leishmania donovani is a major public health problem in the developing world. The emergence of increasing number of L. donovani strains resistance to antimonial drugs recommended worldwide requires the intervention of effective vaccine strategy for treatment of VL. In the present study L. donovani culture derived, soluble, secretory serine protease (pSP) has been shown to be vaccine target of VL. Protection from VL could be achieved by the use of safer vaccine which generally requires an adjuvant for induction of strong Th1 response. To assess the safety, immunogenicity and efficacy of pSP as vaccine candidate in mouse model we used IL-12 as adjuvant. BALB/c mice immunized with pSP+IL-12 were protected significantly from challenged infection even after four months by reducing the parasite load in liver and spleen and suppressed the development of the disease along with an increase in IgG2a antibody level in serum, enhanced delayed type hypersensitivity and strong T-cell proliferation. Groups receiving pSP+IL-12 had an augmented pSP antigen specific Th1 cytokines like IFN- and TNF- response with concomitant decrease of Th2 cytokines IL-4 and IL-10 after vaccination. In this study the vaccine efficacy of pSP was further assessed for its prophylactic potential by enumerating matrix metalloprotease-9 (MMP-9) profile which has been implicated in various diseases. MMP-9 associated with different microbial infections is controlled by their natural inhibitors (TIMPS) and by some cytokines. In this study pSP was found to regulate excessive inflammation by modulating the balance between MMP-9 and TIMP-1 expression. This modulatory effect has also been demonstrated by IFN- mediated down regulation of TNF- induced MMP-9 expression in activated murine macrophages. This is the first report where a secretory L. donovani serine protease (pSP) adjuvanted with IL-12 could also act as protective imunogen by modifying cytokine mediated MMP-9 expression in experimental VL. These findings elucidate the mechanisms of regulation of MMP-9 following infection of L. donovani in vaccinated animals and thus pave the way for developing new immunotherapeutic interventions for VL. © 2012 Elsevier GmbH. All rights reserved. Introduction Leishmaniasis is a vector-borne complex protozoan infection caused by protozoan parasite of the genus Leishmania, which is present in two forms in their life cycle: the promastigotes, that multiplies in the midgut of the sand fly vector, and amastigotes, Abbreviations: CMI, cell mediated immunity; CL, cutaneous leishmaniasis; IFN- , interferon gamma; IL, interleukin; ECM, extra cellular matrix; FCA, Freund’s complete adjuvant; FIA, Freund’s incomplete adjuvant; MMP, matrix metallopro- teinase; pSP, purified serine protease; p.i, post infection; TNF-, tumor necrosis factor-; TIMP, tissue inhibitor metalloproteinase; VL, visceral leishmaniasis. ∗ Corresponding author. Tel.: +91 9239403709; fax: +91 33 25828282. E-mail address: t chakra08@rediffmail.com (T. Chakraborti). 1 Present address: Department of Molecular Medicine and Haematology, Univer- sity of the Witwatersrand, Johannesburg, South Africa. the obligate intracellular form lives within the macrophages of the vertebrate hosts (Bates 2007). The major clinical presentations depend upon the causative species and immunological status of the host. These range from a simple cutaneous lesion through to the disfiguring mucocutaneous leishmaniasis and finally to the vis- ceralized form or kala-azar, which is fatal if left untreated (Murray et al. 2005). The World Health Organization considers leishma- niasis to be one of the most serious epidemics prone parasitic infectious diseases occurring mostly in poor and disadvantaged resulting in substantial morbidity and mortality in an estimated 12 million people worldwide (Desjeux 2004). The control strate- gies for VL mainly rely on vector control and the treatment options with the use of pentavalent antimonials as the first line drugs (Croft 1988). The available chemotherapy for VL is far from satisfactory because antileishmanial drugs are few and costly with severe side effects. Moreover, the current increase in drug resistance (Sundar 0171-2985/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. doi:10.1016/j.imbio.2012.02.008