Major immunodominant proteins of Leptospira interrogans Icterohaemorrahagiae were cloned, expressed in prokaryotic system was purified and confirmed by western blot. The protective efficacy of purified recombinant proteins were assayed synergistically with adjuvant in hamster models. Three combination were assessed for homologous protection (i.e, rLipL41, rOmpL1, rHapI and rSphH; rLipL41, rOmpL1, rLigA and rLigB; rLigA, rLigB, rHapI and rSphH) each protein at 25 µg and 37.5 µg concentration. Among the combinations tested combination of immunogloblulins (rLigA and rLigB) with hemolysins (rHapI and rSphH) confered 100% protection in both the concentration tested, that is evident through the elevated elicited antibody levels. The combination of outer membrane proteins and the immunoglobulins stands next which is comparable with the former combination. The blend of recombinant outermembrane proteins and hemolysins did effect protection which is lesser than the coalescence of immunoglobulins and hemolysins. Membrane fractions of E. coli harboring pET15b used as internal control showed neither homologous protection nor enhanced antibody levels used to compare the experimental proteins over hamster models. Placebo controls receiving PBS showed similar results as that of internal controls. Key words : Leptospiral recombinant protein, recombinant proteins, outer membrane proteins, hemolysins, immunoglobulin like proteins, immune response, challenge studies, , homology modeling REFERENCES: Alves, V.A., Gayotto, L.C., Brito, T.De., Santos, R.T., Wakamatsu, A., Vianna, M.R. and Sakata, E.E. (1992). Leptospiral antigens in the liver of experimentally infected guinea pig and their relation to the morphogenesis of liver damage. Exp. Toxicol. Pathol. 44(7): 425-434. Artiushin, S., Timoney, J.F., Nally, J. and Verma. A. (2004). Host inducibile immunogenic Sphingomyelinases like protein, Lk73.5, of Leptospira interrogans. Infect. Immun. (72): 742-749. Bartlett, J.M.S. and Stirling, D. (2004). DNA Extraction form Fungi, Yeast and Bacteria. PCR Protocols Second Edition, Methods in Molecular Biology, Humana Press, (226): 53-54. Bernheimer , A.W., and Bey, R.F. 1986. Copurification of Leptospira interrogans serovar pomona hemolysin and Sphingomyelinase C. Infect. Immun. 54(1): 262-264. Bolin CA, Cassells, J.A., Zuerner, R.L. and Trueba, G. (1991). Effect of vaccination with a monovalent Leptospira interrogans serovar hardjo type hardjo-bovis vaccine on type hardjo-bovis infection of cattle. Am. J. Vet. Res. (52): 1639-1643. Branger, C., Sonrier, C., Chatrenet, B., Klonjkowski, B. and Ruvoen-Clouet, N. (2001). Identification of the hemolysis-associated protein I as a cross-protective immunogen of Leptospira interrogans by adenovirus-mediated vaccination. Infect. Immun. (69): 6831-6838. Branger, C., Chatrenet, B., Gauvrit, A., Aviat, F. and Aubert, A. (2005). Protection against Leptospira interrogans sensu lato challenge by DNA immunization with the gene encoding hemolysin-associated protein I. Infect. Immun. (73): 4062-4069. Carvalho, E., Barbosa, A.S, Gomez, R.M., Oliveira, M.L.S., Romero, E.C., Goncales, A.P., Morais, Z.M., Vasconcellos, S.A. and Ho, P.L. (2010). Evaluation of the Expression and Protective Potential of Leptospiral Sphingomyelinases. Curr. Microbiol. (60): 134-142. Choy, H.A., Kelley, M.M., Chen, T.L., Moller,A.K., Matsunaga, J. and Haake, D.A. (2007). Physiological osmotic induction of Leptospira interrogans adhesion: LigA and LigB bind extracellular matrix proteins and fibrinogen. Infect. Immun. (75): 2441-2450. Coutinho, M.L., Choy, H.A., Kelley, M.M., Matsunaga, J., Babbitt, J.T., Lewis, M.S., Aleixo, J.G. and Haake, D.A. (2011). A LigA three domain region protects hamsters from lethal infection by Leptospira interrogans. PLoS Negl. Trop. Dis. 5(12): e1422. Porika Raju and Estari Mamidala (2015). Anti-diabetic activity of compound isolated from Physalis angulata fruit extracts in alloxan induced diabetic rats. The Ame J Sci & Med Res, 2015,1(1); Pages 1 -6. doi:10.17812/ajsmr2015.11.1. Croda, J., Ramos, J.G.R., Matsunaga, J., Queiroz, A., Homma, A., Riley, L.W., Haake, D.A., Reis, M.G. and Ko, A.I. (2007). Leptospira immunoglobulin like proteins as a serodiagnostic marker for acute Leptospirosis. J. Clin. Microbiol. 45(5): 1528-1534. Croda, J., Figueira, C.P., Wunder Jr., A.E., Santos, C.S., Reis, M.G., Ko, A.I. and Picardeau, M. (2008). Targeted Mutagenesis in pathogenic Leptospira species: Disruption of the LigB gene does not affect virulence in animal models of leptospirosis. Infect. Immun. 76(12): 5826-5833. Faine, S. (1994). Leptospira and leptospirosis, 1st ed. CRC press, Boca Raton, Fla. Faucher, J.F, Hoen, B. and Estavoyer, J.M. (2004). The management of Leptospirosis. ExpertOpin. Pharmacother. 5(4): 819-827.. Goldstein, S.F. and Charon, N.W. (1990). Multiple exposure photographic analysis of motile spirochetes. Proc Natl Acad Sci, (87): 4895-4899. Haake, D.A., Mazel, M.K., McCoy, A.M., Milward, F., Chao, G., Matsunga, J. and Wager, E.A., (1999). Leptospiral outer membrane proteins OmpL1 and LipL41 exhibit synergistic immune protection. Infect Immun. (67): 6572-6582. Isogai, E., Isogai, H., Kurebayashi, Y. and Ito, N. (1986). Biological activities of Leptospiral lipopolysaccharide. Zentralbl. Bakteriol. Mikrobiol. Hyg. A. (261): 53-64. Koizumi, N. and Watanabe, H. (2004). Leptopsira immunoglobulin-like protein elicit protective immunity. Vaccine (22): 1545-1552. Lee, S.H., Sangduk, K., Seung, C.P. and Min, J.K. (2002). Cytotoxic Activities of Leptospira interrogans Hemolysin SphH as a Pore-Forming Protein on Mammalian Cells. Infect. Immun. (70): 315-322. Levett, P.N. (2001). Leptospirosis. Clin. Microbiol. Rev. (14): 296-326. Lourdault, K., Wang, L.C., Vieira, A., Matsunaga, J., Melo, R., Lewis, M.S., Haake, D.A. and Solecki, M.G. (2014). Oral immunization with Escherichia coli expressing a lapidated form of LigA protects hamsters against challenge with Leptospira interrogans serovar copenhageni. Infect. Immun. 82(2): 893-902. Lucas, D.S., Cullen, P.A., Lo, M., Srikram, A. and Sermswan, R.W. (2011). Recombinant LipL32 and LigA from Leptospira are unable to stimulate protective immunity against leptospirosis in the hamster model. Vaccine. (29): 3413-3418. Matsunaga, J., Barocchi, M.A., Croda, J., Young, T.A., Sanchez, Y., Siqueira, I., Bolin, C.A., Reis, M.G., Riley, L.W., Haake, D.A. and Ko, A.I. 2003. Pathogenic Leptospira species express surface exposed proteins belonging to the bacterial immunoglobulin superfamily. Mol. Microbiol. (49): 929-945. Matsunaga, J., Werneid, K., Zuerner, R.L., Frank, A. and Haake, D.A. (2006). LipL46 is a novel surface exposed lipoprotein expressed during Leptospiral dissemination in the mammalian host. Microbiology. (152): 3777-3786. Merino, S., Rubires, X., Knoche, S. and Tomas, J.M. (1995). Emerging pathogens: Aeromonas spp. Int. J. Food. Microbiol. (28): 157-168. Natarajaseenivasan, K., Shanmughapriya, S., Velineni, S., Artiushin, S.C. and Timoney, J.F. (2011). Cloning, Expression, and Homology Modeling of GroEL Protein from Leptospira interrogans Serovar Autumnalis Strain N2. Genomics Proteomics Bioinform. 9(4-5): 151. Palaniappan, R.U., Chang, Y.F., Jusuf, S.S.D., Artiushin, S., Timoney, J.F., McDonough, S.P., Barr, S.C., Divers, T.J., Simpson, K.W., McDonough, P.L. and Mohammed, H.O. (2002). Cloning and Molecular Characterization of an Immunogenic LigA Protein of Leptospira interrogans. Infect. Immun. 70(11): 5924-5930. Palaniappan, R.U., Chang, Y.F., Hassan, F., McDonough, S.P., Pough, M., Barr, S.C., Simpson, K.W., Mohammed, H.O., Shin, S., McDonough, P.L., Zuerner, R.L., Qu, J. and Roe, B. 2004. Expression of Leptospiral immunoglobulin like protein by Leptospira interrogans and evaluation of its diagnostic potential in a kinetic ELISA. J. Med. Microbiol. (53): 975-984. Palaniappan, R.U., McDonough, S.P., Divers, T.J., Chen, C.S., Pan, M.J., Matsumoto, M. and Chang, Y.F. (2006). Immunoprotection of recombinant Leptospiral immunoglobulin like protein A against Leptospira interrogans serovar Pomona infection. Infect. Immun. (74(3)): 1745-1750. Parthiban, M., Senthil Kumar, S., Balachandran, C., Kumanan, K., Aarthi, K.S. and Nireesha, G. 2015. Comparison of Immunoprotection of Leptospira recombinant proteins with conventional vaccine in experimental animals. Ind. J. Exp. Biol. (53): 779-785. Patti, J.M., Allen, B.L., McGavin, M.J. and Hook, M. (1994). MSCRAMM-mediated adherence of microorganisms to host tissues. Ann. Rev. Microbiol. (48): 585-617. Picardeau, M.D., Bulach, D.M. and Bouchier., C. (2008). Genome sequence of the saprophyte Leptospira biflexa provides insightsinto the evolution of Leptospira and the pathogenesis of leptospirosis. PLoS ONE 3: 1607 Pinnie, M. and Haake, D.A. (2009). A comprehensive approach to identification of surface-exposed, outer membrane spanning proteins of Leptospira interrogans. PLoS One (4): e6071. Seixas, F.K., da Silva, E.F., Hartwig, D.D., Cerqueira, G.M., Amaral, M. (2007). Recombinant Mycobacterium bovis BCG expressing the LipL32 antigen of Leptospira interrogans protects hamster from challenge. Vaccine. 26: 88-95. Silva, E.F., Medeiros, M.A., McBride, A.J., Matsunaga, J., Esteves, G.S., Ramos, J.G., Santos, C.S., Croda, J., Homma, A. and Dellgostin, O.A. (2007). The terminal portion of Leptospiral immunoglobulin-like protein LigA confers protective immunity against lethal infection in the hamster model of leptospirosis. Vaccine. 25(33): 6277-6286. Trowbridge, A.A., Green, J.B., Bonnet, J.D., Shohet, S.B., Ponnappa, B.D. and Mccombs, W.B. 1981. Hemolytic anemia associated with leptospirosis morphologic and lipid studies. Am. J. Clin. Pathol. (76): 493-498. Zuerner, R.L., Alt, D.P., Palmer, M.V., Thacker, T.C. and Olsen, S.C. (2011). A Leptospira borgpetersenii serovar Hardjo vaccine induces a Th1 response, activates NK cells, and reduces renal colonization. Clin. Vaccine. Immunol. (18): 684-691.