The accomplishments of the section are: 1. We discovered a new biological function of a salivary protein, named D7, from the saliva of sand flies. Despite the the notion that blood feeders from independent evolutionary lineages utilize different protein families for various salivary functions, the saliva of sand flies contains apparent homologs of the two-domain long-form D7 proteins of mosquitoes. The similarity is particularly strong in the predicted N-terminal domain of the protein where key amino acids lining the leukotriene/thromboxane binding pocket that interact with the ligand are conserved. We demonstrated, using binding studies and bioassays, that the sand fly long-form D7s do in fact bind both cysteinyl leukotrienes and thromboxanes, and thereby serve as anti-inflammatory and anti-hemostatic agents in both sandflies and mosquitoes. We also solved the crystal structure of the sand fly protein where we show the conserved N-terminal domain as well as the more divergent C-terminal domain and domain interface region related with the mosquito D7 forms and the evolutionary origins of the group as a whole. 2. We identified two sand fly salivary proteins as biomarkers for human exposure to the sand fly Phlebotomus orientalis, a vector of visceral Leishmaniasis in Eastern Africa. Recombinant proteins derived from sequence data on P. orientalis secreted salivary proteins cDNA library were produced using mammalian expression systems HEK293 cells and tested as antigens applicable for detection of anti-P. orientalis IgG in human sera. Human sera from Sudan and Ethiopia, countries endemic for visceral leishmaniasis, were screened by ELISA and immunoblotting to identify the potential markers of exposure to P. orientalis bites. Two recombinant proteins; mAG5 and mYEL1, were identified as the most promising antigens showing high correlation coefficients as well as good specificity in comparison to the whole sand fly salivary gland homogenate. Combination of both proteins led to a further increase of correlation coefficients as well as both positive and negative predictive values of P. orientalis exposure. 3. We demonstrated that co-infection with the parasite Trypanosoma brucei confers protection against cutanoues leishmaniasis. Infection with certain bacteria, parasites, and viruses alters the host immune system to Leishmania major influencing disease outcome. We determined the outcome of a chronic infection with Trypanosoma brucei brucei on cutaneous leishmaniasis (CL) caused by L. major. C57BL/6 mice infected with T. b. brucei were given a sub-curative treatment with diminazene aceturate then coinfected with L. major by vector bites. Our results revealed that infection with T. b. brucei controls CL pathology. Compared to controls, coinfected mice showed a significant decrease in lesion size up to 6 weeks post-infection and a significant decrease in parasite burden at 3 weeks post-infection. Protection against L. major resulted from a non-specific activation of T cells by trypanosomes. This induced a strong immune response characterized by IFN- production at the site of bites and systemically, creating a hostile inflammatory environment for L. major parasites and conferring protection from CL. 4. We identified a sand fly salivary vaccine candidate from the sand fly Phlebotomus sergenti. We tested the immune response in BALB/c mice to 14 different plasmids coding for the most abundant salivary proteins of Ph. sergenti. The plasmid coding for the salivary protein PsSP9 induced a DTH response in the presence of a significant increase of IFN- expression in draining lymph nodes (dLN) as compared to control plasmid and no detectable PsSP9 antibody response. Animals immunized with whole Ph. sergenti SGH developed only a saliva-specific antibody response and no DTH response. Mice immunized with whole Ph. sergenti saliva and challenged intradermally with L. tropica plus Ph. sergenti SGH in their ears, exhibited no protective effect. In contrast, PsSP9-immunized mice showed protection against L. tropica infection resulting in a reduction in nodule size, disease burden and parasite burden compared to controls. These results suggest that this family of proteins in Ph. sergenti, Ph. duboscqi and Ph. papatasi may have similar immunogenic and protective properties against different Leishmania species. Indeed, this anti-saliva immunity may act as an adjuvant to accelerate the cell-mediated immune response to co-administered Leishmania antigens, or even cause the activation of infected macrophages to remove parasites more efficiently. These findings highlight the idea of applying arthropod saliva components in vaccination approaches for diseases caused by vector-borne pathogens. 5. We demonstrated that saliva of the sand fly Lutzomyia longipalpis induces the expression of Hemooxygenase-1 in the skin of animals. Sand flies bite mammalian hosts to obtain a blood meal, driving changes in the host inflammatory response that support the establishment of Leishmania infection. This effect is partially attributed to components of sand fly saliva, which are able to recruit and activate leukocytes. Our group has shown that heme oxygenase-1 (HO-1) favors Leishmania survival in infected cells by reducing inflammatory responses. Here, we show that exposure to sand fly bites is associated with induction of HO-1 in vivo. Histopathological analyses of skin specimens from human volunteers experimentally exposed to sand fly bites revealed that HO-1 and Nrf2 are produced at bite sites in the skin. These results were recapitulated in mice ears injected with a salivary gland sonicate (SGS) or exposed to sand fly bites, indicating that vector saliva may be a key factor in triggering HO-1 expression. Resident skin macrophages were the main source HO-1 at 24-48 h after bites. Additionally, assays in vivo after bites and in vitro after stimulation with saliva both demonstrated that HO-1 production by macrophages was Nrf2-dependent. Collectively, our data demonstrates that vector saliva induces early HO-1 production at the bite sites, representing a major event associated with establishment of naturally-transmitted Leishmania infections. 6. We developed a human protocol to study the immune response to mosquito bites and dengue cases in humans in Cambodia. Mosquito-borne arboviruses, like dengue virus, continue to cause significant global morbidity and mortality, particularly in Southeast Asia. Cumulative evidence from animal models and limited data from humans have identified various vector-derived components, including salivary components, that are co-delivered with the pathogen and play an important role in the dissemination of infection. Much about the roles and effects of these vector-derived factors remain to be discovered. We developed a longitudinal, pagoda (community)-based pediatric cohort study to evaluate the burden of dengue virus infection and document the immune responses to salivary proteins of Aedes aegypti, the mosquito vector of dengue, Zika, and chikungunya viruses. The study includes community-based seroprevalence assessments in the peri-urban town of Chbar Mon in Kampong Speu Province, Cambodia. The study aims to recruit 771 children between the ages of 2 and 9 years for a three year period of longitudinal follow-up, including twice per year (rainy and dry season) serosurveillance for dengue seroconversion and Ae. aegypti salivary gland homogenate antibody intensity determinations by ELISA assays. Diagnostic tests for acute dengue, Zika and chikungunya viral infections will be performed by RT-PCR. This study will serve as a foundation for further understanding of mosquito saliva immunity and its impact on Aedes-transmitted arboviral diseases endemic to Cambodia.
Showing the most recent 10 out of 104 publications
