Chitinases of pathogens have been proposed as potential targets of vaccines or specific inhibitors. We studied the genomic organization, transcript levels, developmental expression, and biological function of chitinases in the rodent filarial nematode Acanthocheilonema viteae, a model organism for human-pathogenic filarial worms. Characterization of nine genomic clones from an A. viteae phage library and Southern blot experiments revealed the existence of three different chitinase genes, two of which could theoretically yield functional transcripts. The deduced proteins of these genes had the common modular organization of family 18 chitinases. Northern blot experiments and rapid amplification of cDNA ends-PCR with adult worms and larval stages showed that only one gene is expressed, with high variation in transcript levels, as determined by real-time PCR. Chitinase transcript levels were lowest in the late male stage 4 larva (L4) and peaked in the stage 3 larva (L3), which was corroborated by Western blotting. RNA interference (RNAi) experiments showed that treatment of L3 and adult female worms with double-stranded RNA of chitinase inhibited molting of L3 worms and hatching of microfilariae. RNAi also led to the death of 50% of female worms, revealing the essential role of chitinase in the life cycle of filarial nematodes.Filarial nematodes are the causative agents of filariases, tropical diseases that afflict about 160 million people worldwide (27). Currently, there are neither safe and efficient drugs nor vaccines available to eliminate or prevent these infections, which makes the development of new control strategies a priority. Chitin, one of the most abundant polysaccharides in nature, is known to be present in the eggshell (7,14,26) and the microfilarial sheath (9) of nematodes and is an integral part of their pharynx (24, 30). Because chitin has not been found in vertebrates, enzymes associated with chitin metabolism might lend themselves as targets for the development of antihelminthic drugs and vaccines.Chitin is metabolized consecutively by two types of glycoside hydrolases, as follows: chitinase breaks down the ␤-1,4-glycosidic bonds of chitin to release N-acetylglucosamine dimers, which are then acted upon by N-acetylglucosaminidase. Enzymes of the glycosyltransferase family are responsible for chitin synthesis.Chitinases, in particular, have been proposed to have a role in remodeling processes during the molting of filariae and in the hatching of larvae from the eggshell (3, 5). However, the existence of large families of chitinases in the free-living nematode Caenorhabditis elegans suggests that these enzymes also fulfill other functions (17). The genome of this nematode contains 42 predicted glycoside hydrolase genes, comprising chitinase and N-acetylglucosaminidase genes (Carbohydrate-Active Enzymes server at http://afmb.cnrs-mrs.fr/CAZY). This raises questions regarding the large number of chitinases in nematodes and what particular roles these enzymes play in the worm's life cycle.Filarial ch...

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Subjects with generalized onchocerciasis (GEN), with the sowdah form, and with exposure but without onchocerciasis (endemic normal/putatively immune; EN/PI) were studied for cytokine responses to Onchocerca volvulus extract (OvAg) and recombinant Ov33 and OvL3-1 proteins. Higher levels of cytokines were produced in response to OvAgs in sowdah and EN/PI than in GEN subjects. Peripheral blood mononuclear cells did not produce interferon-gamma in response to antigens. OvAg induced interleukin (IL)-5, IL-2, granulocyte-macrophage colony-stimulating factor (GM-CSF), and soluble IL-2 receptor. EN/PI and sowdah persons produced significantly more IL-5 and IL-2 than GEN subjects, and EN/PI subjects had significantly higher GM-CSF levels than GEN persons. The low IL-5 and GM-CSF levels in GEN subjects were increased by addition of exogenous IL-2. Ov33 and OvL3-1 stimulated production of IL-10 and less IL-5 and IL-2. The study groups did not show a strict Th2-like cytokine response.

Abstract. Onchocerciasis remains a major health hazard in many tropical countries. However, the existing tools for diagnosis of the disease have limitations, particularly regarding the detection of low level or early infections. To design an optimized reagent, we exploited the high antibody reactivity of patient sera against the Onchocerca volvulus proteins Ov20 and Ov33, which have been described as highly sensitive and specific immunodiagnostic reagents for producing hybrid proteins. The construct OvH2 was composed of Ov20 fused to Ov33, while OvH3 consisted of the C-terminus of Ov20 linked to Ov33. When these constructs were tested with sera from patients with onchocerciasis and control sera, OvH2 showed a sensitivity of 98.5% and a specificity of 97.7% and OvH3 showed a sensitivity of 98.5% and a specificity of 95.35%. All non-responders were from Ecuador. These results surpass those of existing single recombinant antigens, suggesting that our hybrid proteins combined the sensitivity of the two parent proteins. Tests based on OvH2 should prove suitable for monitoring onchocerciasis control programs and individual diagnosis.

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