Using phage display libraries (from infective larvae L3 and adult females) Brugia malayi and screens with known soluble human receptors, we have identified, cloned and characterized distinct molecules that bind to the human IL-5R, IL-10R, and IL-13R. The molecule we term, Bm-IL5RBP Brugia malayi IL5Rbinding protein(IL5Rbp) has been expressed at high levels in a manner that retains its ability to bind to the human receptor. Antibodies raised to predicted immunogenic peptides inhibit the binding of rBm-IL5BP to its human receptor and have been used to localize (by immuno-EM) Bm-IL5RBP to the surface of the infective stage larvae. The rBm-IL5BP does not itself prolong the survival of human eosinophils, but does inhibit the ability of human IL5 to prolong eosinophil survival. ? B. malayi HSP12.6 (BmHSP12.6)has been identified and characterized as a molecule that binds to the human IL-10R. Structural analyses of BmHSP12.6 showed that it has a highly conserved alpha-crystallin central domain that is characteristic of other small heat shock proteins (HSPs) but because of . short N-terminal domain and an unusually small C-terminal domain flanking the crystallin domain appears to belong to a novel class of small HSPs. Recombinant BmHSP12.6 binds to huIL10R in a dose dependent fashion and inhibits the binding of human IL-10 (huIL10) to its receptor. rBmHSP12.6 also enhanced the growth and proliferation of MC/9 mast cells in vitro similar to huIL10, suggesting that this parasite actively synthesizes a moleclule that may modulate the human immune response.? Interestingly, we have also identified a secreted product from Brugia malayi L3 that is chemotactic for human eosinophils. This secreted products signals through the G-coupled receptor CXCR3.? ? We used a molecular approach to show definitively that the fialrial parasite, Mansonella perstans (Mp) contains the endosymbiont, Wolbachia. Using primers known to amplify the 16S ribosomal DNA of other filarial Wolbachiae, an identical 1393bp band was found that on sequence analysis demonstrated a single consensus sequence for Mp Wolbachia 16S rDNA that was most similar to Wolbachia sequences from other filarial nematodes.Phylogenetic dendrograms, examining the relationship of the Mp Wolbachia to other Wolbachia 16S rDNA, showed that the Wolbachia tracked almost identically to the 5S rRNA of their parasite host. Wolbachia surface protein (WSP) was also demonstrated in protein extracted from Mp-containing whole blood. In advance of a treatment trial of Mp, a method for the quantitation of Mp Wolbachia was developed and used to demonstrate not only a relationship between microfilarial numbers and Wolbachia copy numbers, but also to demonstrate the effect of antibiotic on ridding Mp of Wolbachia.? ? As part of a consortium of many workers, we have been a part of the annotation of the filarial genome published in draft from this past year.? ? A separate proteomic analysis has also been completed using mass spectrospcopy in tandem with whole stage-specific tryptic digestion. Not only has the stage specific proteomes been completed for adult males, adult females, microfilariae, and infective larvae, but we have also completed the Brugia Wolbachia proteome and the excretory/secretory proteome. These analyses have provided clear evidence that those genes encoding """"""""hypothetical proteins"""""""" are real and have also identified close to 70% of all predicted open reading frames. Further the secretome analyses have revealed unexpected secreted proteins many of which have host-immunomodulatory properties.? ? An expanded approach to protective immunity in both filarial infections and in strongyloides is underway. Antigens affinity purified using sera from either mice immunized with irradiated larvae that show close to 100% protection to challenge infection with S. strongyloides or humans demonstrating immunity to Ss infection have identified 6 candidates that have been expressed in E. coli, yeast and baculovirus. Testing with one E. coli-derived recombinant has provided 50% protection in a mouse model.? ? Rapid diagnostics have been developed for Strongyloides, Onchocerca and Loa loa infections using mutiplexed luciferase immunoprecitation assays (LIPS). Because Loa loa infections are in need of a more quantitative diagnostic, identification of new diagnostic targets have been undertaken by constructing new Loa cDNA libraries and performing EST generation. 5 candidate diagnostics have been identified and cloned into vectors suitable for LIPS.
Showing the most recent 10 out of 12 publications
