Molecular Definition Of Filarial And Related Nonfilarial Genes And Proteins

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Using an EST (expressed sequence tag) approach, we have been able to identify greater than 965 gene clusters from Wuchereria bancrofti microfilariae based on1687 ESTs. Interestingly, a surprisingly high percentage (584/965; 60%) of these clustered ESTs contain a putative signal peptide, and only 6 appear to be GPI anchored. When compared to Brugia malayi (Bm) mf ESTs/Clusters, 45% of the Wb clusters had matches to Bm ESTs (identities ranging from 86-100%) whereas fewer (22%) matched Onchocerca volvulus (Ov) mf ESTs (with identities ranging from 80-100%). Using a phage display L3 cDNA library of Brugia malayi and screens with known 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 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, 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. 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. As part of a consortium of many workers we have been part of the annotation of the filarial genome, a draft of which will be published in Sept, 2007. 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 (still underway) 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. An expanded approach to protective immunity in both filarial infections and in strongyloides is underway. ha 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 are being expressed in E. coli, yeast and baculovirus to determine which product can be made in large quantities to allow for protein based immunization experiments.