Granuloma formation around filarial larvae triggered by host responses to an excretory/secretory antigen.

In previous studies using a murine model of filarial infection, granuloma formation was found to be a most important host-protective mechanism. We have also shown that in vitro cytoadherence is a surrogate for the formation of antifilarial granulomas in vivo and that it requires "alternatively activated" host cells and a source of antifilarial antibody. We show here that antibodies against L3 excretory/secretory (E/S) products can facilitate in vitro cytoadherence. We generated a set of hybridomas reactive with filarial E/S products and screened them for their ability to mediate in vitro cytoadherence. One clone (no. 1E9) was positive in this assay. We then screened a novel expression library of filarial antigens displayed on the surface of T7 bacteriophage for reactivity with 1E9. Phage expressing two filarial antigens (TCTP and BmALT-2) reacted with 1E9. Immunization of mice showed that the cohort immunized with BmALT-2 cleared a challenge infection with infective Brugia pahangi L3 in an accelerated manner, whereas cohorts immunized with TCTP cleared larvae with the same kinetics as in unimmunized mice. These data confirm that BmALT-2 is the antigenic target of granuloma-mediated killing of B. pahangi L3. Our findings also confirm previous studies that BmALT-2 is a potential vaccine candidate for filarial infection. Our data reinforce the work of others and also provide a possible mechanism by which immune responses to BmALT-2 may provide host protection.

Hsp90 is essential in the filarial nematode Brugia pahangi.

The development of a compound with activity against filarial nematodes (a 'macrofilaricide') has been a long-standing goal of the World Health Organization. However, adult filariae have proved remarkably difficult to kill. To some extent this reflects a lack of understanding of key pathways and processes in filarial nematodes that may be suitable targets for chemotherapy. In this paper we show that geldanamycin (GA), a specific inhibitor of the activity of the heat shock protein 90 (Hsp90) family, kills adult worms and microfilariae (Mf) of Brugia pahangi at nanomolar concentrations. In addition, release of Mf from adult worms is inhibited within 24 h of exposure to GA and is not recoverable, demonstrating that GA effectively sterilises the worm. Similar results were obtained with a second filarial worm Acanthocheilonema viteae. In contrast GA has no effect on the free-living nematode Caenorhabditis elegans despite a high degree of conservation between the nematode Hsp90 sequences. In keeping with these findings, Brugia Hsp90 binds GA in a solid phase pull-down assay while the binding of C. elegans Hsp90 to immobilised GA is undetectable. In other eukaryotes, GA is known to bind in the N-terminal ATP pocket of Hsp90, disrupting its interactions with client proteins which are then targeted for degradation via the proteasome pathway. Thus, Hsp90 or some of its client proteins may provide novel targets for the chemotherapy of filarial infection.

The ultrastructure of the gravid uterus of Brugia pahangi, another rich source of antigen of the filarial parasite.

The gravid uterus with zygotes and microfilariae in utero of Brugia pahangi, a rich source of antigen as revealed by a recent immunofluorescent technique, were studied ultrastructurally. The epithelial cells of uterus show ultrastructural features of synthetically active cells. Their secretions may provide nutrients for the egg in utero. On the basal side, the uterine epithelial cells may also secrete substances to form the basal lamina of the uterus which is rather thick and irregularly fused with the basal lamina lining the body wall where the pseudocoelomic cavity is obliterated. For the most part, the uterine basal lamina contains uniform granular material of moderate electron density. There are also elongated visceral muscle cells embeded in it, and which surround the uterus, with adjacent cells overlapping. The gravid uterus contains several stages of developing microfilariae within its lumen, the cleaving zygotes are also present at another level. The morula of zygotes are composed of several closely packed cells surrounded loosely by their own egg shell membranes. The egg shell becomes more convoluted as development proceeds. The egg shell surrounding the developing microfilariae in utero is secreted by the uterine epithelium. This structure later becomes the sheath of circulating microfilariae, and is highly antigenic as indicated by intense labeling with fluorescent antibodies.