The accomplishments of the section are: 1. We solved the structure of the salivary vaccine candidate SALO and implemented an expression and purification plan to move this vaccine candidate forward to the clinical path. Having selected LJM19 (SALO) as one of our lead vaccine candidates based on the results from the clinical study in humans and from efficacy studies in rodents, we developed a plan for its expression and to study the characteristics of this protein. Recombinant SALO was expressed as a soluble secreted protein using Pichia pastoris. Unlike its native counterpart, rSALO(P) does not inhibit the classical pathway of complement; however, antibodies to rSALO(P) inhibit the anti-complement activity of sand fly salivary gland homogenate. Immunization with rSALO(P) produces a delayed type hypersensitivity response in C57BL/6 mice, suggesting rSALO(P) lacked anti-complement activity but retained its immunogenicity. The structure of rSALO(P) was solved by S-SAD at Cu-Kalpha to 1.94 . SALO is 80% helical, has no appreciable structural similarities to any human protein, and has limited structural similarity in the C-terminus to members of insect odorant binding proteins. The results indicate that SALO as expressed and purified from P. pastoris is suitable for further scale-up, manufacturing, and testing. 2. We characterized the sand fly gut microbiome and studies the consequences of the insect microbiota in Leishmania development. In collaboration with Mary Wilson from Iowa State University, we characterized the sand fly midgut microbiome and studied the effect of the microbiome on Leishmania development. In this study, the microbiota within the sand fly Lu. longipalpis midgut was delineated by 16S ribosomal DNA (rDNA) sequencing, revealing a highly diverse community. Interestingly, infection with Leishmania parasites resulted in loss of microbial diversity that was most pronounced in mature infections. Importantly, removing the sand fly gut microbiota in the early stages of parasite development with an antibiotic cocktail, which alone had no effect on either the parasite or the fly, halted development of the Leishmania parasites and abrogated subsequent development to the infective form. Together, these data suggest that the sand fly midgut microbiome is a critical factor for Leishmania development and growth inside the sand fly. 3. We identified a sand fly salivary protein as specific marker for Lutzomyia intermedia exposure Sand flies inject saliva while feeding in the vertebrate host and anti-saliva antibodies can be used as biomarkers of exposure to Leishmania vectors. We expressed recombinant salivary proteins from Lutzomyia intermedia, a vector of Leishmania braziliensis, and evaluated the seroreactivity in exposed individuals in search for exposure markers. We found a strong correlation among positive serology to recombinant proteins LinB-13, 26, 15, 21 and to salivary proteins: rLinB-13 was the top performing molecule; IgG4 was the most predominant antibody subclass and antibodies to rLinB-13 did not cross react with Lu. longipalpis salivary proteins. By evaluating a cohort of contacts of CL patients, we confirmed that rLinB-13, an antigen 5-related protein, is a marker of exposure to Lu. intermedia with high degree of accuracy. In a 5-year follow up, we determined that individuals who developed CL presented higher anti-rLinB13 IgG responses, before the appearance of clinical symptoms. They also presented a lower frequency of cellular responses to the parasite (DTH). Our results show that seroconversion to a salivary molecule, rLinB-13, is a marker of risk for CL development caused by Leishmania braziliensis. This highlight the possibility of developing tools based on vector molecules to manage the disease in endemic areas.
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