The Vector Molecular Biology Unit focuses on the molecular aspects of sand fly salivary and midgut proteins with emphasis on the understanding of vector/host and vector/parasite interactions, specifically sand fly/Leishmania interactions. Unit research combines basic approaches together with veterinary and clinical research broadening our understanding of the relationship between immune responses to vector proteins in animal reservoirs and humans and disease outcome, and between the Leishmania parasite and the sand fly midgut proteins to ultimately develop a vector-based vaccine against the neglected disease leishmaniasis. 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 acceleration of anti-Leishmania immunity. 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 accomplishment from the two main projects of the unit are: ? ? ? 1) Development of a robust protein recombinant expression and purification methodology to test salivary proteins for biological activities, and validate vaccine candidates identified using DNA immunization. ? We maximized expression of a properly folded recombinant salivary protein by cloning sand fly salivary gland transcripts into the mammalian expression vector developed by this unit, VR2001-TOPO, the same vector used for DNA immunization, and used it to transfect 293F mammalian cells. Transfected cells produced soluble recombinant proteins and by following an optimized purification methodology we obtained a number of salivary proteins in large quantities, highly pure and with a minimal amount of endotoxin for testing in vaccine, immunological and biological assays. ? ? 2) The demonstration that a recombinant sand fly salivary protein protects against challenge with L. major infected sand flies. ? We demonstrated that the protection observed by immunization with DNA vaccine is not a bystander effect of the DNA plasmid and it is specific to the protein encoded by the plasmid. We tested the protective effect of recombinant protein LJM11 that is encoded in the selected DNA plasmid against the virulent challenge of L. major infected Lu. longipalpis sand fly bites. Mice immunized with a small amount of recombinant LJM11 in the absence of adjuvants were protected against challenge by bites from ten infected sand flies (Figure 4). This is the first demonstration of the protective role of a recombinant sand fly salivary protein against cutaneous leishmaniasis and validates the use of DNA immunization for protection against Leishmania infections. ? ? 3) The finding that immunity to distinct sand fly salivary proteins primes the anti-Leishmania immune response towards protection or exacerbation of disease.? Two distinct DTH-inducing salivary proteins from P. papatasi were investigated for their effect on L. major infection. DNA immunization with these molecules resulted in contrasting outcomes of infection upon challenge with L. major parasites. PpSP15-immunized mice showed lasting protection while immunization with PpSP44 aggravated the infection (without an increase in parasite load as compared to the control group). This suggests that immunization with these distinct molecules alters the course of anti-Leishmania immunity. Two weeks post-infection 31.5% of CD4+ T cells produced IFN- in PpSP15-mice compared to 7.1% in PpSP44-mice. IL-4 producing cells were three-fold higher in PpSP44-mice. Two hours after challenge with SGH and L. major, the expression profile of PpSP15-mice showed over three-fold higher IFN- and IL-12-R2 and 20-fold lower IL-4 expression, relative to PpSP44-mice suggesting that salivary proteins differentially prime anti-Leishmania immunity. This demonstrates for the first time that immunity to a salivary protein (PpSP44) results in disease enhancement and stresses the importance of the proper selection of vector-based vaccine candidates. ? ? ? 4) The finding that immunity to a salivary protein of a sand fly vector protects against the fatal outcome of visceral leishmaniasis in a hamster model.? Two distinct DTH-inducing salivary proteins from P. papatasi were investigated for their effect on L. major infection. DNA immunization with these molecules resulted in contrasting outcomes of infection upon challenge with L. major parasites. PpSP15-immunized mice showed lasting protection while immunization with PpSP44 aggravated the infection (without an increase in parasite load as compared to the control group). This suggests that immunization with these distinct molecules alters the course of anti-Leishmania immunity. In this work, we demonstrated that not all DTH-inducing P. papatasi sand fly salivary molecules are universally protective against L. major infection and that immunization with two DTH-inducing salivary proteins produced distinct immune profiles that correlated with resistance or susceptibility to Leishmania infection. This demonstrates for the first time that immunity to a salivary protein (PpSP44) results in disease enhancement and stresses the importance of the proper selection of vector-based vaccine candidates. ? ? 5) Identification of biological activities from novel salivary molecules of unknown sequence or function.? Most sand fly salivary proteins, including the identified protective molecules, are novel and do not have an assigned biological function. Soluble recombinant proteins were produced in a mammalian expression system to test potential biological activities. We identified a potent anticoagulant salivary protein from Lu. longipalpis that binds specifically the active site of factor Xa. We also identified four salivary recombinant proteins that inhibited the alternate pathway of complement by binding specifically to C3b and one protein that inhibits the classical pathway of complement. These molecules have the potential for use in the treatment of a variety of human ailments. This work not only demonstrates that the anticoagulant inhibitor of sand flies is a novel prothrombinase inhibitor, but also makes it a potential active drug to correct pro-coagulant disorders in human medicine. C3b-inhibitors could be potential new drugs that correct human diseases related to complement such as auto-immune diseases and post-surgery complications due to complement. ? ? 6) Identification of the most abundant midgut transcripts of Lutzomyia longipalpis, the vector of Leishmania infantum chagasi.? We have generated and sequenced five cDNA libraries from the midgut tissue of Lu. longipalpis; including midgut tissue after a sugar meal, a blood meal and a blood meal in the presence of L. infantum chagasi. Combining all cDNA library sequences produced 655 contigs, 2279 singletons and an average of 9.45 sequences per contig with approximately 10,000 sequences in total. This transcriptome analysis represents the largest set of sequence data reported from a specific sand fly tissue and provides further information of the transcripts present in the gut of the sand fly Lu. longipalpis.
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