Evaluation of different ways of presenting LipL32 to the immune system with the aim of developing a recombinant vaccine against leptospirosis Fabiana Ko ¨ mmling Seixas, Claudia Hartleben Fernandes, Daiane Drawanz Hartwig, Fabricio Rochedo Conceic ¸a ˜ o, Jose ´ Anto ˆ nio Guimara ˜ es Aleixo, and Odir Anto ˆ nio Dellagostin Abstract: Leptospirosis, caused by bacteria of the genus Leptospira, is a direct zoonosis with wide geographical distribu- tion. The implications in terms of public health and the economical losses caused by leptospirosis justify the use of a vac- cine against Leptospira in human or animal populations at risk. In this study, we used the external membrane protein LipL32 as a model antigen, as it is highly immunogenic. The LipL32 coding sequence was cloned into several expression vectors: (i) pTarget, to create a DNA vaccine; (ii) pUS973, pUS974, and pUS977 for expression in BCG (rBCG); and (iii) pAE, to express the recombinant protein in Escherichia coli, for a subunit vaccine. Mice were immunized with the various constructs, and the immune response was evaluated. The highest humoral immune response was elicited by the subunit vaccine (rLipL32). However, with rBCG, the titer was still rising at the end of the experiment. The serum of vaccinated animals was able to recognize LipL32 on the membrane of the Leptospira, detected by indirect immunofluorescence. A monoclonal antibody anti-LipL32 was shown to inhibit the growth of Leptospira in vitro, indicating potential protection in- duced by the LipL32 antigen. Key words: Leptospira, LipL32, recombinant BCG, subunit vaccine, DNA vaccine. Re ´sume ´: La leptospirose, cause ´e par des bacte ´ries du genre Leptospira, est une zoonose directe dont la distribution ge ´o- graphique est vaste. Les implications en termes de sante ´ des populations et de perte e ´conomique cause ´e par la leptospirose justifient l’utilisation d’un vaccin dirige ´ contre Leptospira chez l’humain ou la population animale a ` haut risque. Dans cette e ´tude, nous avons utilise ´ la prote ´ine LipL32 de la membrane externe de la bacte ´rie comme antige `ne mode `le car elle est hautement immunoge `ne. La se ´quence codante de LipL32 a e ´te ´ clone ´e dans plusieurs vecteurs d’expression : (i) pTar- get, pour cre ´er un vaccin a ` ADN; (ii) les vecteurs pUS973, pUS974 et pUS977 pour permettre l’expression dans la BCG (rBCG); et (iii) pAE pour exprimer la prote ´ine recombinante chez Escherichia coli dans la perspective d’un vaccin a ` sous- unite ´. Les souris ont e ´te ´ immunise ´es avec les diffe ´rentes constructions et la re ´ponse immune a e ´te ´e ´value ´e. La re ´ponse im- mune humorale la plus forte a e ´te ´ obtenue avec le vaccin a ` sous-unite ´ (rLipL32). Cependant le titre du rBCG continuait d’augmenter a ` la fin de l’expe ´rience. Le se ´rum des animaux vaccine ´s e ´tait capable de reconnaı ˆtre LipL32 membranaire de Leptospira, tel que de ´montre ´ par immunofluorescence. Un anticorps monoclonal anti-LipL32 a pu inhiber la croissance de Leptospira in vitro, indiquant une protection potentielle induite par l’antige `ne LipL32. Mots-cle ´s : Leptospira, LipL32, BCG recombinant, vaccin a ` sous-unite ´, vaccin a ` ADN. [Traduit par la Re ´daction] Introduction Leptospirosis, a worldwide zoonotic infection with a high incidence rate in tropical regions, is classified as an emerg- ing infectious disease (McBride et al. 2005). Transmission to humans occurs either through direct contact with an in- fected animal or through indirect contact via soil or water contaminated with urine from an infected animal (Faine 1982). There are more than 230 serovars among pathogenic leptospiras. The local variability in serovars of endemic lep- Received 19 September 2006. Revision received 24 November 2006. Accepted 27 November 2006. Published on the NRC Research Press Web site at cjm.nrc.ca on 25 May 2007. F.K. Seixas and D.D. Hartwig. Centro de Biotecnologia, Universidade Federal de Pelotas, C.P. 354, 96010-900, Pelotas, Brazil. C.H. Fernandes and F.R. Conceic ¸a ˜o. Centro de Biotecnologia, Universidade Federal de Pelotas, C.P. 354, 96010-900, Pelotas, Brazil; Faculdade de Veterina ´ria, Universidade Federal de Pelotas, C.P. 354, 96010-900, Pelotas, Brazil. J.A.G. Aleixo. Centro de Biotecnologia, Universidade Federal de Pelotas, C.P. 354, 96010-900, Pelotas, Brazil; Faculdade de Nutric ¸a ˜o, Universidade Federal de Pelotas, C.P. 354, 96010-900, Pelotas, Brazil. O.A. Dellagostin. 1 Centro de Biotecnologia, Universidade Federal de Pelotas, C.P. 354, 96010-900, Pelotas, Brazil; Instituto de Biologia. Universidade Federal de Pelotas, C.P. 354, 96010-900, Pelotas, Brazil. 1 Corresponding author (e-mail: odirad@terra.com.br). 472 Can. J. Microbiol. 53: 472–479 (2007) doi:10.1139/W06-138 # 2007 NRC Canada