Molecular ID Of Filarial/Nonfilarial Genes/Proteins

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Abstract

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-IL5BP [Brugia malayi IL5 binding 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-IL5BP 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 Using the first generation Brugia malayi expression microarrays, we have been able (not surprisingly) to identify a large number of adult male- and adult female-specific transcripts and corroborate their expression using quantative RT-PCR (Ndi et al, unpublished). We await the second generation microarray (containing >12000 oligos) before proceeding with specific experiments using valuable L3 and other filarial RNA. A similar approach has been taken with Strongyloides stercoralis with >10000 ESTs being done from two separate lifecylcle stages. Comparisons between this parasitic nematode and the free-living C. elegans has been perfomed; the data demonstrate the vast differences in gene expresssion between parasitic and non-parasitic nematodes (Mitreva, DM, McCarter, JP. Marin, J, Dante, M, Wylie, T, Chipelli, B, Pape, D, Clifton, SW. Nutman, TB, Waterston, RH. Comparative Genomics of Gene Expression in the Parasitic and Free-living Nematodes Strongyloides stercoralis and Caenorhabditis elegans. Genome Res 14: 209-220, 2004). A separate proteomic approach to protective immunity is being performed in parallel to the genomic approach. 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 are being analyzed using 2D-PAGE coupled to mass spectroscopy and/or internal protein sequence analyses. Among those proteins identified, four have been characterized and cloned (Ss-eat-6, Ss-tpn-1, Ss-xxx, Ss-xxx). One, Ss-eat-6 has been shown to induce modest protection in mice vaccinated with a DNA vaccine containing Ss-eat-6