Department of Pathobiology, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran. Introduction and Objectives: The worldwide incidence of leptospirosis is increasing year on year, from an initial estimate of 500,000 cases in 1999 to over one million cases and 60,000 fatalities in 2015. Leptospirosis is caused by numerous distinct serovars of a spiral-shaped bacterium known as Leptospira interrogans and it is a disease of animals and humans. These serovars are harbored by a wide range of animals, and all of them are capable of causing illness in humans. Inactivated whole-cell vaccines (bacterins) are routinely used in livestock and domestic animals, however, protection is serovar-restricted and short-term only. To overcome these limitations, efforts have focused on the development of recombinant vaccines, with partial success and recently, reverse vaccinology (RV) has been successfully applied to Leptospirosis for prospective vaccine antigens. Materials and Methods: The most abundant protein in the entire leptospiral proteome is an outer membrane lipoprotein of 32 kDa, LipL32, accounting for 75% of the outer membrane proteome. LipL32 is involved in the protective response against L. interrogans serovar canicola in hamsters and is the first reported link to LipL32- induced protection against kidney invasion. The most promising results regarding protection against leptospirosis were achieved using the leptospiral immunoglobulin-like (Lig) B, A proteins. Reverse vaccinology (RV) has been widely used for screening of surface-exposed proteins (PSEs) of important pathogens, including outer membrane proteins (OMPs), and extracellular proteins (ECPs) as potential vaccine candidates. Results: It has been shown that LipL32 had significantly higher survival rates (P < 0.05) than animals from the control group. This is the first report of a protective immune response afforded by a subunit vaccine using LipL32.While there are reports that LigB can induce protection in animal models, LigA induced an unequivocal immune protection in the hamster model using recombinant protein immunization. However, the ligA gene is present only in three Leptospira spp. (L. interrogans, L. kirschneri and L. alstonii), making it difficult for a LigA vaccine to broadly protect against leptospirosis. As hundreds of draft genomes of all known Leptospira species are now available, this should aid novel target discovery through reverse vaccinology (RV) and pangenomic studies. The identification of surface-exposed vaccine candidates that are highly conserved among infectious Leptospira spp. is a requirement for the development of a cross-protective universal vaccine. Conclusions: The urgent need for a new vaccine has motivated several research groups to evaluate the protective immune response induced by recombinant vaccines. Significant protection has been reported with several promising outer membrane proteins, including LipL32 and the leptospiral immunoglobulin-like proteins. However, efficacy was variable and failed to induce a cross-protective response or sterile immunity among vaccinated animals. As a large number of annotated proteins in Leptospira genomes does not have any known orthologues, RV represents the most promising approach for the discovery of a recombinant vaccine, thereby reducing the burden of leptospirosis.

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