Avanços no estabelecimento de bases para o desenvolvimento de vacinas recombinantes contra a leptospirose

Abstract

Leptospirosis is a zoonosis with worldwide incidence that is caused by pathogenic leptospires. The in vitro growth of pathogenic Leptospira spp. remains difficult and can be a time-consuming process. An important contribution to the microbiology of the genus Leptospira was the development of the EMJH culture medium (EllinghausenMcCullough-Johnson-Harris), which enabled the cultivation of several fastidious strains and became a widely used medium in the field. However, knowledge of the influence of in vitro growth on leptospiral virulence is limited, resulting in altered virulence that can negatively influence the evaluation of vaccine candidates. Recombinant vaccines have been studied as promising alternatives for the control of leptospirosis, however only a few antigens have shown potential as universal vaccine candidates. New approaches in the discovery phase, including reverse vacinology (RV) and structural vacinology (SV), cell surface immunoprecipitation (CSIP) and the use of host-adapted leptospires have been used to identify novel proteins. These approaches identified differentially expressed antigens with immunogenic potential, and which were predicted to be exposed on the cell surface. This thesis includes four manuscripts on the challenges faced in studies using recombinant vaccines against leptospirosis. A published literature review highlighting these topics in the research for a human recombinant vaccine is also included. The in vitro cultivation of L. interrogans pathogenic strains Fiocruz L1-130 and RCA and the L. kirschneri 61H strain was used to compare two different EMJH compositions (commercial and in-house), at 28°C and 37°C, using three different flasks. Subsequently, the impact of in vitro growth conditions on virulence was evaluated via the 50% lethal dose (LD50) in the hamster model of leptospirosis. Both the IHM and commercial EMJH media, incubated at 28°C, in static tubes provided the best growth conditions. For the isolation of leptospires from clinical samples, the IHM offered a significant improvement compared to commercial EMJH. The lowest initial inoculum for IHM was one leptospira compared to 1000 leptospires for commercial EMJH when grown under the following conditions: 28°C and static tubes. Leptospires in the exponential growth phase (early, mid and late) were highly virulent (LD50 ranged from one to 13 leptospires). While the virulence of leptospires in the stationary phase was significantly reduced by >250 fold (LD50 >1000 leptospires). These results allowed us to develop an optimized protocol for the in vitro cultivation of virulent leptospires.. The Fiocruz L1-130 strain was used to produced host-adpated leptospires by the DMC technique followed by CSIP (using antibodies in human sera from convalescent leptospirosis patients)to identify novel leptospiral proteins. CSIP identified 24 immunogenic proteins that were predicted to be localized on the leptospiral outer membrane. Of these, the immunoprotective potential of LICs 10496, 10881 and 11086 were evaluated in the hamster model of acute leptospirosis. The vaccine targets were immunogenic by Western blotting, however they did not induce protective immune responses against leptospirosis. In summary, the results obtained represent advances in the search for recombinant vaccines against leptospirosis and should contribute towards the evaluation of vaccine candidates in13 animal models of leptospirosis. Permitting the comparison of results from different research laboratories, a major limitation at present.