The Vector Molecular Biology Unit focuses on the molecular aspects of arthropod vector salivary and midgut proteins in vector/host and vector/parasite interactions. The two main hypotheses driving the research of this unit are: 1) Cellular immune responses to vector arthropod salivary proteins produce an inhospitable environment in the skin of the host to the co injected pathogen, resulting in indirect killing or unsuccessful establishment in the vertebrate host. Identifying the vector salivary proteins and the correlates of protection particularly the initial immunological events will help us to understand the immunologic basis of protection and to select vaccine candidates to prevent pathogen transmission. 2) Specific molecular interactions between the sand fly midgut and the Leishmania parasite are required for Leishmania survival and development to the infective stage in the insect vector. Characterization of these molecular interactions will help in the understanding of the molecular basis of Leishmania sand fly interactions and may identify a suitable target for a transmission blocking vaccine. The efforts of the Unit are directed toward developing functional genomic approaches based on high throughput DNA vaccine construction, DNA immunization strategies, reverse antigen screening, immunologicl assays, and biochemical approaches to characterizing the interactions between vector and mammalian host and vectors and the parasite they carry. Additionally, these vector proteins may represent potential vaccine candidates to control vector borne diseases. The unit has successfully developed a comprehensive approach to isolate and characterize salivary proteins from different sand flies. With this approach we have performed phylogenetic studies and identified sand fly genus specific and species specific salivary proteins. Additionally, we identified the common proteins between different sand fly genera. Furthermore, we have developed a functional genomic approach to identify vector salivary proteins that can produce antibody and or cellular immune responses which may be detrimental for pathogen or parasite establishment. The approach is based in the selection of transcripts coding for secreted proteins from a sand fly salivary gland cDNA library, cloning of these molecules into a high-throughput DNA vaccine plasmid, and intradermal immunization of these DNA plasmids in animals to test for production of immune responses. With this approach we identified vector salivary proteins capable to produce a delayed skin response (DSR), a surrogate of cellular immune response. With this approach we identified sand fly salivary DNA plasmids that can produce DSR and prevent cutaneous leishmaniasis in mice. The unit has also increased the capabilities to colonize different sand flies in our insectary facilities and we are currently developing more natural approaches to infect sand flies with leishmania parasites in order to test the recently developed DNA vaccines in our unit. Using a comprehensive approach based DNA sequencing of high quality sand fly midgut cDNA library, the unit has identified a number of midgut molecules that are relevant for vector/parasite interaction. One of these molecules was PpChit1 which encodes a midgut specific chitinase. PpChit1 is detected only in the midgut and is regulated by blood feeding. A recombinant protein (rPpChit1) produced in HEK 293-F cells exhibited a similar activity profile to that found in the native protein against several specific substrates, including an oligomeric glycol chitin and synthetic 4-methyl-umbelliferone labelled substrates. Western blotting showed that the native protein is recognized by mouse polyclonal antibodies against rPpChit1. Additionally, the rPpChit1 and the native chitinase displayed similar retention times in a HPLC size fractionation column. When added to rPpChit1 or to midgut lysates, PpChit1 sera reduced chitinolytic activity by 65-70%. The unit has also identified a galactose binding protein (galectin) in the sand fly midgut as the receptor for the Leishmania parasite. This receptor is essential for Leishmania parasite survival and development in the gut of the fly. The practical use of this knowledge is the development of transmission blocking vaccines which target vector midgut proteins and interrupt the attachment of the parasite to the sand fly midgut hence preventing development of the parasite in the insect gut. These findings demonstrate that vector midgut proteins are potential targets for transmission blocking vaccines.
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