The accomplishments of the section are: 1. We have characterized of the early inflammatory infiltrate at the feeding site of infected sand flies in mice protected from vector-transmitted Leishmania major by exposure to uninfected bites. Sand flies transmit Leishmania parasites during blood feeding. Salivary molecules are deposited alongside parasites and can reshape the host's immune response to infection. Exposure to uninfected sand fly bites or immunization with salivary molecules protects the host against Leishmania infection. In this work mice were characterized the formerly unknown early cellular infiltrate at the bite site following L.major vector-transmission. The kinetics and nature of the inflammatory response at the bite site of exposed mice were notably different from those of nave mice showing an amplified expression of cytokines and chemokines after parasite transmission. The transcripts reflected a faster and more robust infiltrate of immune cells to the bite site of exposed mice composed of neutrophils, macrophages, monocytes, NK cells and CD4+ T cells. In addition, there was an increased influx of activated IFN-γproducing CD4+ T cells and Granzyme B-producing mature NK cells in exposed animals. These findings suggest that the observed robust and persistent proinflammatory response in exposed animals restrict parasite multiplication. 2. We identified and characterized a Sand Fly Salivary Endonuclease named Lundep, that Increases Leishmania Parasite Survival in Neutrophils and Inhibits XIIa Contact Activation in Human Plasma Neutrophils are the host's first line of defense against infections, and their extracellular traps (NET) were recently shown to kill Leishmania parasites. Here we report a NET-destroying molecule, named Lundep, from the salivary glands of the sand fly Lutzomyia longipalpis. Lundep is a highly active ss- and dsDNAse, with little sequence specificity with a calculated DNase activity of 300000 Kunitz units per mg of protein. Disruption of PMA (phorbol 12 myristate 13 acetate)- or parasite-induced NETs by treatment with recombinant Lundep increases parasite survival in neutrophils. Furthermore, co-injection of recombinant Lundep with metacyclic promastigotes significantly exacerbates Leishmania infection in mice when compared with PBS alone or inactive Lundep. We hypothesize that Lundep helps the parasite to establish an infection by allowing it to escape from the leishmanicidal activity of NETs early after inoculation. Lundep may also assist blood meal intake by lowering the local viscosity caused by the release of host DNA and as an anticoagulant by inhibiting the intrinsic pathway of coagulation. 3. We achieved an Enhanced protective efficacy of the nonpathogenic recombinant leishmania tarentolae expressing cysteine proteinases when combined with the sand fly salivary antigen PpSP15. Live attenuated vaccines are the gold standard for protection against intracellular pathogens such as Leishmania and there have been new developments in this field. The lizard protozoan parasite, L. tarentolae, is nonpathogenic to humans and has been used effectively as a vaccine platform against visceral leishmaniasis in experimental animal models. Correspondingly, pre-exposure to sand fly saliva or immunization with salivary proteins has been shown to protect mice against cutaneous leishmaniasis. In this work we used DNA/Live and Live/Live prime-boost vaccination strategies against cutaneous leishmaniasis based on recombinant L. tarentolae stably expressing CPA/CPB genes with and without the sand fly salivary antigen PpSP15 in both resistant and susceptible mice models. Assessment of the immune response and parasite burden in vaccinated mice at different time intervals post-challenge demonstrated that combination of recombinant L. tarentolae CPA/CPB with PpSP15 DNA elicits an enhanced protective immune response against cutaneous leishmaniasis in mice. This parasite- and insect vector-derived antigen combination represents an important step forward in the development of new vaccine strategies against Leishmania infections. 4. A Comparative Analysis of Salivary Gland Transcriptomes of Phlebotomus orientalis Sand Flies from Endemic and Non-endemic Foci of Visceral Leishmaniasis. Phlebotomus orientalis is the vector of visceral leishmaniasis (VL) caused by Leishmania donovani in Northeast Africa. In this study, we characterized the salivary compounds of P. orientalis, thereby broadening the repertoire of salivary proteins of sand fly species belonging to the subgenus Larroussius. In order to find out whether there is any connection between the composition of P. orientalis saliva and the epidemiology of VL in two distinct Ethiopian foci, Addis Zemen and Melka Werer, we studied the transcriptomes, proteomes, enzymatic activities, and the main salivary antigens in two P. orientalis colonies originating from these areas. We did not detect any significant difference between the saliva of female sand flies originating in Addis Zemen (endemic area) and Melka Werer (non-endemic area). Therefore, the different epidemiology of VL in these Ethiopian foci cannot be related to the distant salivary gland protein composition. Identifying the sand fly salivary gland compounds will be useful for future research focused on characterizing suitable salivary proteins as potential anti-Leishmania vaccine candidates. 5. The Identification and characterization of Kv1.3 channel-blocking immunomodulatory peptides from parasitic worms. The voltage-gated potassium (Kv) 1.3 channel is widely regarded as a therapeutic target for immunomodulation in autoimmune diseases. ShK-186, a selective inhibitor of Kv1.3 channels, ameliorates autoimmune diseases in rodent models, and human phase 1 trials of this agent in healthy volunteers have been completed. In this work, we identified and characterized a large family of Stichodactyla helianthus toxin (ShK)-related peptides in parasitic worms. Based on phylogenetic analysis, 2 worm peptides were selected for study: AcK1, a 51-residue peptide expressed in the anterior secretory glands of the dog-infecting hookworm Ancylostoma caninum and the human-infecting hookworm Ancylostoma ceylanicum, and BmK1, the C-terminal domain of a metalloprotease from the filarial worm Brugia malayi. These peptides in solution adopt helical structures closely resembling that of ShK. At doses in the nanomolar-micromolar range, they block native Kv1.3 in human T cells and cloned Kv1.3 stably expressed in L929 mouse fibroblasts. They preferentially suppress the proliferation of rat CCR7- effector memory T cells without affecting naive and central memory subsets and inhibit the delayed-type hypersensitivity (DTH) response caused by skin-homing effector memory T cells in rats. Further, they suppress IFNγproduction by human T lymphocytes. ShK-related peptides in parasitic worms may contribute to the potential beneficial effects of probiotic parasitic worm therapy in human autoimmune diseases.
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