Avaliação da atividade imunogênica de três proteínas de Leptospira interrogans expressas em Escherichia coli.

SOUZA, N. M. Evaluation of immunogenic activity of three proteins of Leptospira interrogans expressed in Escherichia coli. 2013. 140 p. Masters thesis (Biotechnology)Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, 2013. Leptospirosis is a zoonosis with great impact in public health of developing countries. The disease is caused by pathogenic bacteria of the genus Leptospira, and is transmitted by urine of infected animals. In the world, 500,000 cases are reported each year with a mortality rate of 10%. However, these data are possibly underestimated due to the difficulties of diagnosis. The symptoms are unspecific, ranging from flu-like symptoms to severe clinical cases with pulmonary, renal and hepatic failure (Weil syndrome). The control of leptospirosis is difficult to implement. Vaccines against leptospirosis are composed by killed whole cells and have many drawbacks, such as low efficacy, the requirement for annual booster immunizations, and the failure to confer cross-protective immunity against different serovars. In the genome sequencing of Leptospira interrogans serovar Copenhageni, putative outer membrane proteins that might be involved in pathogenicity were identified. After genome analysis, based on cellular localization prediction, the genes LIC11121, LIC11087, LIC11228 and LIC11084 were chosen and the immunogenicity of the corresponding proteins was evaluated. Proteins coded by the genes LIC11087, LIC11228 and LIC11084 were probably located in outer membrane of L. interrogans, as assessed by immunofluorescence microscopy. These genes were amplified by specific primers and cloned in the expression vector pAE. The correct cloning was checked by DNA sequencing. The proteins were expressed in Escherichia coli BL21 SI and E. coli BL21 (DE3), and were further purified by metal chelating chromatography. Mice were immunized and produced antibodies against recombinant proteins. Moreover, the protein rLIC11084 induced the secretion of IFNγ and promoted lymphoproliferation in one of two independent experiments. This protein induced a protective response in 58% of immunized hamsters against lethal challenge. However, this protection was not statistically significant. The proteins rLIC11087, rLIC11228 and rLIC11084 were recognized by antibodies in confirmed serum samples of patients at the convalescent phase of the disease. In addition, the protein rLIC11087 was detected by antibodies in serum of hamsters experimentally infected. The results may suggest expression of these proteins during infection.The proteins rLIC11087 and rLIC11228 bind laminin and plasma fibronectin. These interactions may be important for adhesion of the bacteria to extracellular matrix components during tissue colonization. rLIC11087, rLIC11228 and rLIC11084 interact with plasminogen and could be converted to the enzymatically active plasmin. This may have implications in mechanism of degradation of extracellular matrix and capacity of evasion of immune system. The protein rLIC11087 also interacts with C4bp and fibrinogen and may have a role in immune evasion and inhibition of fibrin clot formation.