Identification of Novel Leishmania donovani Antigens that Help Define Correlates of Vaccine-Mediated Protection in Visceral Leishmaniasis Sudipta Bhowmick, Nahid Ali* Infectious Diseases and Immunology Division, Indian Institute of Chemical Biology, Kolkata, West Bengal, India Abstract Visceral leishmaniasis (VL), caused by the intracellular parasite Leishmania donovani is a major public health problem in the developing world. But there is no effective and safe vaccine approved for clinical use against any form of leishmaniasis. Through reactivity with kala-azar patient and cured sera, polypeptides ranging from 91 to 31-kDa from L. donovani promastigotes were previously identified as potential protective vaccine candidates. In this study four polypeptides 91(LD91), 72 (LD72), 51(LD51) and 31 (LD31)-kDa were purified using sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by electroelution. We compared the vaccine efficacy of these antigens encapsulated in cationic liposomes in BALB/c mice against challenge infection with L. donovani. Our results demonstrated that liposomal LD31 (74%– 77%) and LD51 (72%–75%) vaccination reduced parasite burden to the greatest degree followed by liposomal LD72 (65%– 67%) and LD91 (46%–49%). Analysis of the cytokine responses in immunized mice revealed that all the vaccinated groups produced prechallenge interferon-c, interleukin-12 and interleukin-4. Interestingly, the degree of reduction in parasite load could be predicted by the magnitude of the cytokine responses which correlated inversely with the parasite burden both in liver and spleen. The 31, 51 and 72-kDa bands were identified as ATP synthase a chain, b-tubulin and heat shock 70-related protein 1 precursor of L. major, respectively using matrix-assisted laser desorption ionization–time of flight (MALDI-TOF/ TOF) mass spectrometry. These three leishmanial antigens have not been described before as successful vaccine candidates examined against in vivo VL model. Thus, these antigens can be potential components of future antileishmaniasis vaccines. Citation: Bhowmick S, Ali N (2009) Identification of Novel Leishmania donovani Antigens that Help Define Correlates of Vaccine-Mediated Protection in Visceral Leishmaniasis. PLoS ONE 4(6): e5820. doi:10.1371/journal.pone.0005820 Editor: Elizabeth Didier, Tulane National Primate Research Center, United States of America Received January 28, 2009; Accepted May 1, 2009; Published June 5, 2009 Copyright: 2009 Bhowmick, Ali. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from the Council of Scientific and Industrial Research and Department of Science and Technology, Government of India. It was performed during the tenure of S.B. as a fellow of the Council of Scientific and Industrial Research. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: nali@iicb.res.in Introduction Leishmaniasis is a vector-transmitted disease distributed world- wide mainly in the tropical and subtropical countries. At least 20 Leishmania species can give rise to a wide spectrum of clinical manifestations, ranging from self-healing cutaneous lesions to visceral leishmaniasis (VL), the latter being an invariably fatal disease in the absence of drug treatment. Moreover, Leishmania is emerging as important opportunistic pathogen in persons coinfected with human immunodeficiency virus. Therapeutic options for controlling leishmaniasis are limited to a few drugs with inconsistent efficacy and side effects. Thus, there is an urgent need to develop a safe and effective Leishmania vaccine to help prevent the two million new cases of leishmaniasis worldwide each year [1]. Recent progress to design vaccines against leishmaniasis is based on molecularly defined antigens, the so-called second-generation vaccines. The availability of the complete L. major and L. infantum genome sequence may lead to identification of genes responsible for the different disease phenotypes [2], but vaccine candidates will probably be harder to pinpoint in the genome because without knowing the function of a pathogen protein its protective potential is not guaranteed [3]. A large number of leishmanial antigens against experimental leishmaniasis have been attempted for vaccination, mainly against the cutaneous form. There are also reports of defined antigens protective against VL in animal models such as KMP11, HASPB, A2, ORFF and CPB [4–8]. However there is still only a little progress and a limited number of potential candidates to combat this disease through vaccination [9]. Moreover, proteins identified from cutaneous form when tested against challenge infection with L. donovani in experimental models have found to be either partially protective or unsuccessful [10– 12]. Thus there is a need for identifying new antigens from L. donovani ideally relying on correlates of protective immunity. Several studies of L. major support that resistance towards infection is linked to the ability to mount a Th1 response which leads to the activation of macrophages for elimination of the obligatory intracellular parasite [13]. Protective immunity in L. donovani is also dependent on an IL-12 driven Th1 and IFN-c production [14,15]. However, an exclusive generation of a vaccine-induced Th1 is insufficient to ensure protection and cannot be a predictor of vaccine success in experimental VL [10,16]. In murine cutaneous leishmaniasis (CL) a Th2 response and IL-4 production have been associated with chronic infection [13]. Although induction of IL-4 in infected BALB/c and noncuring models [17,15] has been observed, beneficial roles of PLoS ONE | www.plosone.org 1 June 2009 | Volume 4 | Issue 6 | e5820