Vaccine 24 (2006) 2900–2909 Leishmania donovani: Identification of stimulatory soluble antigenic proteins using cured human and hamster lymphocytes for their prophylactic potential against visceral leishmaniasis Ravendra Garg a , Shraddha K. Gupta a,1 , Parul Tripathi b,1 , K. Hajela d , S. Sundar c , S. Naik b , Anuradha Dube a,∗ a Division of Parasitology, Central Drug Research Institute, Lucknow, India b Department of Immunology, Sanjay Gandhi Post-Graduate Institute of Medical Sciences, Lucknow, India c Department of Medicine, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India d School of Life Sciences, Devi Ahilya University, Indore, India Received 14 December 2005; accepted 21 December 2005 Available online 17 January 2006 Abstract Most of the studies for the identification of prophylactic antigens that elicit T cell responses were concentrated on membrane proteins of Leishmania donovani. This study was taken up to assess L. donovani soluble promastigote antigens for their ability to stimulate proliferation of peripheral blood mononuclear cells (PBMCs) from cured visceral leishmaniasis (VL) patients, endemic and non-endemic controls and lymphocytes/peritoneal macrophages of cured hamsters. The soluble protein was subjected to sequential precipitation with saturated ammo- nium sulphate (20%, 40%, 60% and 80%), of which largely 80% fractioned protein showed significant cellular responses in cured patients and hamsters. This fraction was further fractionated into five sub fractions by preparative SDS–PAGE and subjected to re-evaluation for their ability to induce cellular responses. Out of these, only F2 sub fraction belonging to the MW of 97.4–68 kDa stimulated remarkable lymphoproliferative and IFN- responses in cured VL patients and in endemic controls. Similarly, significant lymphoproliferative responses and nitric oxide production were also noticed in cured Leishmania infected animals indicating an element of uniformity in responses between hamster and human. F2 sub fraction, when evaluated for its prophylactic efficacy with BCG against L. donovani challenge in hamster exhibited significant parasite inhibition in spleen (71.1%; p < 0.001) and liver (68.2%; p < 0.001) as compared to their unvaccinated counterpart. The vaccinated animals showed significant lymphoproliferative response and nitric oxide production but leishmania specific IgG level were suppressed. The results indicate the presence of immunostimulatory and protective molecules in F2 sub fraction which may further be exploited for the development of a vaccine against VL, hitherto an unrealized goal. © 2005 Elsevier Ltd. All rights reserved. Keywords: Visceral leishmaniasis (VL); Soluble Leishmania donovani promastigote (SLD); T cell stimulatory antigen; Cured VL patients; Cured hamsters; Prophylactic efficacy ∗ Corresponding author. Tel.: +91 522 212411 212418x4398; fax: +91 522 223405/223938. E-mail addresses: anuradha dube@hotmail.com, anuradha dube@rediffmail.com (A. Dube). 1 S.K.G. and P.T. have contributed equally to the work. 1. Introduction Leishmania species are obligatory intracellular protozoan parasites responsible for a wide spectrum of diseases ranging from local self-healing cutaneous disease (CL) to potentially fatal visceral infection of the spleen and liver [1]. The dis- ease is an important global public health problem with an estimated 350 million people at risk of infection [2]. Vis- 0264-410X/$ – see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2005.12.053