Despite several studies describing the secretion of exosomes by Leishmania in vitro, observation of their formation and release in vivo has remained a major challenge. Herein, we show that Leishmania constitutively secretes exosomes within the lumen of the sand fly midgut through a mechanism homologous to the mammalian pathway. Through egestion experiments, we demonstrate that Leishmania exosomes are part of the sand fly inoculum and are co-egested with the parasite during the insect's bite, possibly influencing the host infectious process. Indeed, co-inoculation of mice footpads with L. major plus midgut-isolated or in-vitro-isolated L. major exosomes resulted in a significant increase in footpad swelling. Notably, co-injections produced exacerbated lesions through overinduction of inflammatory cytokines, in particular IL-17a. Our data indicate that Leishmania exosomes are an integral part of the parasite's infectious life cycle, and we propose to add these vesicles to the repertoire of virulence factors associated with vector-transmitted infections.

Sand flies are blood-feeding insects and vectors of the Leishmania parasite. For many years, saliva of these insects has represented a gold mine for the discovery of molecules with anti-hemostatic and immuno-modulatory activities. Furthermore, proteins in sand fly saliva have been shown to be a potential vaccine against leishmaniasis and also markers of vector exposure. A bottleneck to progress in these areas of research has been the identification of molecules responsible for the observed activities and properties of saliva. Over the past decade, rapid advances in transcriptomics and proteomics resulted in the completion of a number of sialomes (salivary gland transcriptomes) and the expression of several recombinant salivary proteins from different species of sand fly vectors. This review will provide readers with a comprehensive update of recent advances in the characterization of these salivary molecules and their biological activities and offer insights pertaining to their protective effect against leishmaniasis and their potential as markers of vector exposure.

Immunity to a sand fly salivary protein protects against visceral leishmaniasis (VL) in hamsters. This protection was associated with the development of cellular immunity in the form of a delayed-type hypersensitivity response and the presence of IFN-γ at the site of sand fly bites. To date, there are no data available regarding the cellular immune response to sand fly saliva in dogs, the main reservoirs of VL in Latin America, and its role in protection from this fatal disease. Two of 35 salivary proteins 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-γ production following in vitro stimulation of PBMC with salivary gland homogenate (SGH). Upon challenge with uninfected or infected flies, immunized dogs developed a cellular response at the bite site characterized by lymphocytic infiltration and IFN-γ and IL-12 expression. Additionally, SGH-stimulated lymphocytes from immunized dogs efficiently killed Leishmania infantum chagasi within autologous macrophages. Certain sand fly salivary proteins are potent immunogens obligatorily co-deposited with Leishmania parasites during transmission. Their inclusion in an anti-Leishmania vaccine would exploit anti-saliva immunity following an infective sand fly bite and set the stage for a protective anti-Leishmania immune response. Leishmaniasis is a neglected infectious disease with a global distribution encompassing 88 countries, 350 million people at risk, and an annual incidence of 2 million cases. Leishmaniasis is a vector-borne disease transmitted by sand fly bites where parasites are co-deposited with saliva into the wound. Our group has demonstrated that distinct molecules in the saliva of various sand fly species drive an immune response that protects experimental rodent models from self-healing cutaneous and fatal visceral leishmaniasis. Here we show for the first time that dogs, natural reservoirs of visceral leishmaniasis, develop a strong immune response to two salivary proteins from the natural vector sand fly. Blood from immunized dogs contained immune cells that produced molecules (IFN-γ) typically associated with protection from Leishmania parasites. This response efficiently recruited appropriate immune cells to the site of sand fly bites in the skin and had an adverse effect on Leishmania parasites in an experimental assay. These findings suggest that inclusion of these salivary molecules in anti-Leishmania canine vaccines would enhance their efficiency in protecting dogs from visceral leishmaniasis. A successful anti-Leishmania canine vaccine would not only protect dogs from a fatal disease but could have a considerable effect on reducing human infections.

Currently, there are no commercially available human vaccines against leishmaniasis. In rodents, cellular immunity to salivary proteins of sand fly vectors is associated to protection against leishmaniasis, making them worthy targets for further exploration as vaccines. We demonstrate that nonhuman primates (NHP) exposed to Phlebotomus duboscqi uninfected sand fly bites or immunized with salivary protein PdSP15 are protected against cutaneous leishmaniasis initiated by infected bites. Uninfected sand fly–exposed and 7 of 10 PdSP15-immunized rhesus macaques displayed a significant reduction in disease and parasite burden compared to controls. Protection correlated to the early appearance of Leishmania-specific CD4⁺IFN-γ⁺ lymphocytes, suggesting that immunity to saliva or PdSP15 augments the host immune response to the parasites while maintaining minimal pathology. Notably, the 30% unprotected PdSP15-immunized NHP developed neither immunity to PdSP15 nor an accelerated Leishmania-specific immunity. Sera and peripheral blood mononuclear cells from individuals naturally exposed to P. duboscqi bites recognized PdSP15, demonstrating its immunogenicity in humans. PdSP15 sequence and structure show no homology to mammalian proteins, further demonstrating its potential as a component of a vaccine for human leishmaniasis.