Using sera from individuals identified as """"""""putatively immune"""""""" to Onchocerca volvulus infections, cDNA expression libraries have been screened; approximately 60 L3 and L4 recombinants have been identified and gridded for counter-screening with sera from well- defined patients with early patent infection. Concurrently, each has had partial sequence obtained (EST analysis and the recombinants have been placed in groups based on their homologies to known proteins in the databases although many have been shown to encode novel proteins). Genes encoding the four most promising (as vaccine targets) have been placed in expression vectors (yeast, bacterial-based expression systems) and the recombinant proteins are being characterized. This includes a filarial 1,6 bis phosphate aldolase that not only induces a strong immune response in animals, but also is a target of T cells in putatively immune individuals. Identifying and characterizing those molecules from the larval stages of the filarial parasites that induce protective immunity in lymphatic filariasis has also remained a large part of our effort. Using novel T-cell-based and antibody-based screening techniques, we have identified 12 vaccine targets from Wuchereria bancrofti larval cDNA expression libraries. The approach will be both traditional (over-expression of recombinant products) and DNA-based using both the proprietary Vical vectors as well as several re-engineered vectors that have shown promise in other systems. The development of diagnostic antigens for lymphatic filariasis has also been undertaken. To this end, a cDNA expression library from adult female parasites was constructed and differentially screened with appropriate sera. More than 25 individual recombinants have been identified as potentially diagnostic, and expression and extensive testing with large panels of well- defined sera has identified four recombinants that appeared to be specific for individuals with lymphatic filariasis. Sequence analysis indicated that 3/4 were interrelated and homologous to HMG-like proteins; one was unique and not homologous to any protein in the various databases. The unique polypeptide, termed Wb1.2 and one of the HMG-like proteins, Wb5.4, were expressed at high levels in several different prokaryotic expression vectors. Partially purified protein was then used as the basis for immunoblot assays using well-characterized sera from individuals with W. bancrofti infection (n=36) and B. malayi infection (n=9) as well as from individuals with nonlymphatic filarial infections (n=27), and nonfilarial parasitic infections (n=36). Normal individuals without parasites (n=15) were also studied. Using these recombinants alone or in combination gave sensitivities that ranged from 65 to 98% and specificities from 89 to 100%. In addition, sets of overlapping peptides have been synthesized and utilized to map the B-cell epitopes of these two proteins in the hopes of utilizing a peptide-based assay for diagnosis of patent filarial infection.
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