Vacinologia reversa e estrutural: identificação da localização celular de alvos vacinais contra leptospirose

Abstract

Leptospirosis is a neglected tropical disease responsible for more than 1 million cases worldwide every year and approximately 60,000 deaths. Spirochetes belonging to the Leptospira genus are the causative agent of leptospirosis. The only vaccine available for disease control is a bacterin that, while protective and extensively used in animals, induces a short-term, serovar restricted, immune response and causes adverse side-effects. There is a need for the development of a new leptospirosis vaccine to control the spread of this disease. In the present work, an innovative approach for vaccine target discovery based on reverse and structural vaccinology is presented. The objective of the current work was to confirm the localization of potential vaccine candidates on the L. interrogans cell surface through the development of a new in vitro technique. Eighteen outer membrane proteins with a β-barrel transmembrane structure were identified and predicted to contain immunogenic epitopes. These proteins were conserved among the infectious Leptospira spp. Nine proteins were expressed in Escherichia coli and used to produce polyclonal antibodies. Following characterization, the antibodies were used to evaluate the localization of the corresponding native proteins using a surface immunofluorescence assay. This assay confirmed that LIC10881 is an L. interrogans surface-exposed β-barrel transmembrane protein. However, the same approach erroneously suggested the flagellar protein FcpA as a surface-exposed outer membrane protein. The approach based on the encapsulation of spirochetes in low melting point agarose microdroplets was adapted to L. interrogans. This assay confirmed that the lipoproteins LigB, LipL32 and LcpA are exposed on the bacterial surface. This is promising approach for the experimental demonstration of surface protein localization in L. interrogans, however it still requires further experimental evaluation.