The accomplishments of the section are: 1. A new model of progressive visceral leishmaniasis in hamsters initiated by natural transmission via bites of vector sand flies. We used Lu. longipalpis to transmit Le. infantum or Le. donovani to hamsters. Vector-transmitted L. infantum and L. donovani caused ≥5-fold increase in spleen weight compared to uninfected organs and had geometric mean parasite loads (GMPL) comparable to the intracardiac inoculation of 107108 parasites, although vector-initiated disease progression was slower with a greater weight loss. Only vector-initiated L. infantum infections caused cutaneous lesions at transmission and distal sites. Importantly, sand flies feeding on ear, mouth, and testicular lesions, respectively, were parasite-positive. Successful transmission was associated with a high mean percent of metacyclics (66%82%) rather than total GMPL (2.0 1048.0 104) per midgut. This model provides an improved platform to study initial immune events at the bite site, parasite tropism, pathogenesis, to test drugs and vaccines against naturally acquired VL. 2. Characterization of the cellular immune response to sand fly salivary proteins in humans living in a P. duboscqi prevalent area in Mali. This study was conducted in the villages of Kemena and Sougoula. During the first 3 years of this study, PBMCs were obtained from 255 individuals aged 1165 years to assess systemic immunity to sand fly saliva. Furthermore, 191 individuals distributed by age groups participated in a study on the DTH response to uninfected sand fly bites. The two cohorts were randomly selected from a population of 1,549 individuals. Peripheral blood mononuclear cells (PBMCs) from 98% of 255 volunteers responded specifically to stimulation with P. duboscqi salivary gland homogenate (SGH). Three subpopulations were distinguished by their systemic immune response to SGH: 23% were polarized toward a TH1 response producing high IFN-γand low IL-13 and IL-5 levels;25% were polarized toward a TH2 response inducing high IL-13 and IL-5 and low IFN-γlevels, and 52% displayed a mixed TH1/TH2 response. These proportions lead us to hypothesize that individuals with a TH1-polarized response are relatively resistant to cutaneous leishmaniasis. On the other hand, individuals with a TH2-polarized response would reflect a proportion of the population that would not be protected from and perhaps are made more susceptible to leishmaniasis by previous exposure to sand fly saliva. 3. Delayed-type hypersensitivity to P. duboscqi salivary proteins is TH1-mediated and persists to midlife. Induction of a TH1 DTH response to sand fly saliva in rodents is strongly associated to protection against leishmaniasis. The nature and potential relevance to leishmaniasis of DTH to sand fly bites in naturally exposed humans remains unknown. Here, we describe the duration and type of DTH to sand fly saliva in humans permanently residing in our study villages. DTH was assessed at 24, 48, 72, and 96 hours post bite in volunteers exposed to colony-bred sand flies. Dermal biopsies were obtained 48 hours post bite. The DTH response to sand fly bites was long-lasting being observed in 75% of individuals aged 115 years, decreasing gradually to 48% by age 45, and dropping to 21% thereafter. Dermal biopsies of positive DTH individuals were dominated by T lymphocytes and macrophages. Abundant expression of IFN-γand absence of TH2 cytokines establishes the TH1 nature of this DTH response. These immune responses are reminiscent of those that protect against leishmaniasis in animal models. It remains to be determined whether this sustained TH1-mediated DTH response confers significant protection against CL in humans as it does in animal models. 4. Characterization of salivary gland transcriptomes from relevant worldwide sand fly vectors. We analyzed the salivary gland transcriptomes of the sand fly Phlebotomus papatasi, Lutzomyia ayacuchensis and Lutzomyia intermedia. Following is an example of one of these transcriptomes. A cDNA library from P. papatasi (Tunisian strain) salivary glands was sequenced and analyzed. The most abundant secreted proteins were categorized as Odorant Binding Proteins/D7 Superfamily, antigen 5-related proteins, apyrases, yellow-related proteins, and PpSP32-like proteins. We also identified proteins not described before in this sand fly species like SP34 belonging to the Lufaxin family of anti-coagulants. To note, comparative analysis between the salivary proteins of P. papatasi from Tunisian and Israeli strains shows a high level of identity. Thus, these proteins represent potential targets as common markers of vector exposure for different geographical areas. Similar analysis was performed with the above-mentioned salivary gland transcriptomes. 5. Salivary antigen SP32 is the immunodominant target of the antibody response to Phlebotomus papatasi bites in humans. We expressed in mammalian cells two salivary proteins PpSP30 and PpSP32. The two recombinant salivary proteins were purified by two-step HPLC and tested to determine which of these proteins correspond to the immunodominant 30kDa antigen previously shown to be recognized by sera of humans from endemic areas naturally exposed to P. papatasi. Recombinant PpSP32 (rPpSP32) was strongly recognized by the tested sera;in contrast, rPpSP30 was poorly recognized. The binding of human IgG antibodies to native PpSP32 was inhibited by the addition of rPpSP32. Our findings demonstrate that PpSP32 is the immunodominant target of the human antibody response to P. papatasi saliva.

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