The accomplishment from the two main projects of the unit are: 1) Discovery of two sand fly salivary vaccine candidates that produce a strong and long lasting cellular immune response in dogs. Dogs are the main reservoir of visceral leishmaniasis in Europe and South America. We developed an experimental model that mimics the natural exposure of dogs to sand fly bites and explored the immune responses to salivary proteins from the sand fly Lutzomyia longipalpis, the vector of visceral leishmaniasis in South America. Lu. longipalpis salivary proteins induced a DTH response at the bite site in the skin of dogs following repeated exposures to sand flies. This skin response is a surrogate of cellular immune response and a first screen marker of protection against Leishmania infection in rodent models. Two of 35 salivary proteins (LJM17 and LJL143) from the vector sand fly Lutzomyia longipalpis, identified using a novel approach termed reverse antigen screening, elicited strong cellular immunity in dogs. Immunization with either molecule induced high IgG2 antibody levels and significant IFN- production following in vitro stimulation of PBMC with salivary gland homogenate. Importantly, saliva stimulated lymphocytes from immunized dogs efficiently killed Leishmania infantum chagasi within autologous macrophages. These findings suggest that inclusion of these salivary molecules in anti-Leishmania canine vaccines would enhance their efficiency in protecting dogs from visceral leishmaniasis. 2) Identification of biological activities from novel salivary molecules of unknown sequence or function. Most sand fly salivary proteins are novel and do not have an assigned biological function. Soluble recombinant proteins were produced in a mammalian expression system to test potential biological activities. We identified a potent serotonin binding protein, a potent anticoagulant, a tryptase inhibitor and an anti-complement protein from the saliva of L. longipalpis. These molecules have the potential for use in the treatment of a variety of human ailments. This work not only demonstrates that the anticoagulant inhibitor of sand flies is a novel prothrombinase inhibitor, but also makes it a potential active drug to correct pro-coagulant disorders in human medicine. C3b-inhibitors could be potential new drugs that correct human diseases related to complement such as auto-immune diseases and post-surgery complications due to complement. 3) Discovery of Markers of Exposure Specific to Bites of Lutzomyia longipalpis, the Vector of Leishmania infantum chagasi in Latin America Leishmania parasites are transmitted by the bite of an infected vector sand fly that injects salivary molecules into the host skin during feeding. Certain salivary molecules can produce antibodies and can be used as an indicator of exposure to a vector sand fly and potentially the disease it transmits. To identify potential specific markers of vector exposure, we produced nine different recombinant salivary proteins from Lu. longipalpis and tested for their recognition by individuals exposed to another human-biting sand fly, Lu. intermedia, that transmits cutaneous leishmaniasis and commonly occurs in the same endemic areas as Lu. longipalpis. Two salivary proteins (named LJM17 and LJM11) were recognized only by humans exposed to Lu. longipalpis, suggesting they are immunogenic proteins and may be useful in epidemiological studies. The identification of specific salivary proteins as potential markers of exposure to vector sand flies will increase our understanding of vector human interaction, bring new insights to vector control, and in some instances act as an indicator for risk of acquiring disease. 4) Characterization of the salivary proteins from the sand fly Phlebotomus arabicus. We performed a transcriptomic analysis of the old world sand fly Phlebotomus arabicus and analyzed the repertoire of molecules in the saliva of this insect, performed bioinformatics analyses and some biological assays to validate the presence of powerful biological activities and antigenic molecules.
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