Tellervo Huima

Cloning of a Cysteine Protease Required for the Molting of Onchocerca volvulus Third Stage Larvae

We have investigated the involvement of a cysteine protease in the development of Onchocerca volvulus fourth stage larvae (L4) by testing the effect of cysteine protease inhibitors on the survival of third stage larvae (L3), and the molting of L3 to L4 in vitro. When larvae were cultured in the presence of specific inhibitors, the peptidyl monofluoromethylketones, viability of either L3 or L4 was not affected. However, the inhibitors reduced the number of L3 that molted to L4 in vitro in a time- and dose-dependent manner. Molting was completely inhibited in the presence of 50-250 μM inhibitor. Ultrastructural examination of L3 that did not molt in the presence of inhibitors indicated that new L4 cuticle was synthesized, but there was no separation between the L3 and the L4 cuticles. The endogenous cysteine protease was detected in molting larvae after binding to labeled inhibitors, and by antibodies directed against a recombinant O. volvulus L3 cysteine protease that was cloned and expressed. The enzyme was detected in cuticle regions where the separation between the cuticles occurs in molting larvae. These studies suggest that molting and successful development of L4 depends on the expression and release of a cysteine protease.

Characterization of an Onchocerca volvulus cDNA clone encoding a genus specific antigen present in infective larvae and adult worms

The isolation and characterization of a recombinant cDNA clone (OV7) expressing an antigen present in Onchocerca volvulus infective larvae and adult stages is described. Using chimpanzee antiserum generated against irradiated infective larvae, we isolated a cDNA clone from a lambda gt11 cDNA expression library derived from adult O. volvulus mRNA. The open reading frame encodes 131 amino acids corresponding to a 15.2-kDa protein. Affinity purified antibodies which bound specifically to OV7 fusion polypeptide recognized a single antigen with an apparent molecular weight of 17,000 in extracts of L3, L4 and adult worms. Immunoelectron microscopy established that the antigen encoded by this clone is present in the hypodermis and the basal layer of the cuticle of L3 and female adult worm, and in the egg shell around developing microfilariae. Since the OV7 fusion polypeptide is onchocerca-specific and is recognized specifically by sera from onchocerciasis patients, and sera from non-patent but infected chimpanzees, and not by sera from patients with other filarial parasites, it may have potential as an antigenic component in a test for detection of non-patent and patent infections of O. volvulus. The OV7 amino acid sequence contains residues that have a probable homology with the cysteine proteinase inhibitor superfamily.

Inactivation of hepatitis B and Hutchinson strain non-A, non-B hepatitis viruses by exposure to Tween 80 and ether.

Titrated stocks of hepatitis B virus and Hutchinson strain non‐A, non‐B hepatitis virus were diluted in normal serum to contain, respectively, ≥106 and ≥104 chimpanzee infectious doses (CID50) per milliliter and exposed to 1% Tween 80 and 20% ether at 4°C for 18 h. After evaporation of the ether, the treated sera were each inoculated into two chimpanzees. The animals remained free of serologic and biochemical evidence of hepatitis during a 6‐month follow‐up period, and were then shown to be susceptible to infection by challenge with the original untreated inocula.

Transglutaminase-catalyzed reaction is important for molting of Onchocerca volvulus third-stage larvae.

Highly insoluble proteins, which are probably cross-linked, are common in the cuticle and epicuticle of filarial parasites and other nematode species. We have investigated the possible involvement of transglutaminase (TGase)-catalyzed reactions in the development of Onchocerca volvulus fourth-stage larvae (L4) by testing the effects of TGase inhibitors on the survival of third-stage larvae (L3) and the molting of L3 to L4 in vitro. The larvae were cultured in the presence of three specific TGase inhibitors: monodansylcadaverine, cystamine, and N-benzyloxycarbonyl-D,L-beta-(3-bromo-4,5-dihydroisoxazol-5-yl)-al anine benzylamide. None of the inhibitors reduced the viability of either L3 or L4. However, the inhibitors reduced, in a time- and dose-dependent manner, the number of L3 that molted to L4 in vitro. Molting was completely inhibited in the presence of 100 to 200 microM inhibitors. Ultrastructural examination of L3 that did not molt in the presence of monodansylcadaverine or cystamine indicated that the new L4 cuticle was synthesized, but there was an incomplete separation between the L3 cuticle and the L4 epicuticle. The product of the TGase-catalyzed reaction was localized in molting L3 to cuticle regions where the separation between the old and new cuticles occurs and in the amphids of L3 by a monoclonal antibody that reacts specifically with the isopeptide epsilon-(gamma-glutamyl)lysine. These studies suggest that molting and successful development of L4 also depends on TGase-catalyzed reactions.

ISOLATION OF A VIRUS FROM CHIMPANZEE LIVER CELL CULTURES INOCULATED WITH SERA CONTAINING THE AGENT OF NON-A, NON-B HEPATITIS

A membrane-coated virus having a diameter of 85-90 nm and containing a 40-45 nm core was found to replicate in cell cultures derived from chimpanzee liver after inoculation of serum containing infective non-A, non-B (NANB) hepatitis viruses from two independent sources. Replication of this agent was not observed when the same cells were inoculated with a chloroform-extracted inoculum or were left uninoculated. Replication involves assembly of virus cores on tubular structures similar to those seen in liver cells of chimpanzees infected with most isolates of NANB virus.

Onchocerca volvulus: Biochemical and morphological characteristics of the surface of third- and fourth-stage larvae

The annulated cuticles of third- and fourth-stage larvae of Onchocerca volvulus have the typical structure of other nematodes but the cuticle of fourth-stage larvae was thinner. The surface of the third-stage larva was wrinkled and fuzzy, while that of the fourth-stage was smooth. Intermediate stages in the formation of the new cuticle and epicuticle beneath the old basal layer and of the separation of the cuticles are shown. Monoclonal antibodies specific to the surface of third-stage larvae did not react with the surface of the fourth-stage larvae. Binding of the monoclonal antibodies to the third-stage larvae was abrogated by treatment of the worms with trypsin and proteinase K, but was unaffected by treatment with periodate or the detergents sodium deoxycholate and SDS. The lectins RCA120 and WGA, but not any of the other lectins tested, bound only to the surface of fourth-stage larvae, and not to that of third-stage larvae. The surfaces of third- and fourth-stage larvae were shown to be different and contained stage-specific surface epitopes.

Identification and characterization of an Onchocerca volvulus cDNA clone encoding a microfilarial surface-associated antigen

The identification and characterization of a recombinant cDNA clone (OV103) expressing a microfilarial surface-associated antigen of Onchocerca volvulus is described. OV103 was identified and isolated from a lambda gt11 cDNA expression library derived from adult O. volvulus mRNA using a chimpanzee antiserum, taken 2 years after infection with third-stage larvae of O. volvulus. The cDNA clone encodes a 12.5-kDa protein that corresponds to a 15-kDa parasite protein present in microfilariae and adult female worms. The antigen encoded by this clone is located in the basal layer of the cuticle and the hypodermis of the female adult worm, and on the surface of microfilariae. OV103 fusion polypeptide is recognized only by some sera from onchocerciasis infected subjects (57%), but more significantly (89%) by sera from individuals that have low levels of patent infection. In addition, the antibody response to this protein developed before appearance of microfilariae in the skin of chimpanzees that had developed non-patent or low level patent infections, while the antibody response in chimpanzees with high levels of microfilariae appeared later at the time of appearance of microfilariae. Preliminary experiments indicated that affinity purified antibodies directed against OV103 fusion polypeptide mediated killing of nodular microfilariae in vitro in the presence of normal peripheral blood granulocytes.

Onchocerca volvulus: characterization of a highly immunogenic Gln-rich protein

A pool of sera from individuals classified as putatively immune (PI) to Onchocerca volvulus infection was employed in the screening of a fourth-stage larval cDNA expression library. A highly immunogenic clone, encoding the Ov 53/80 protein, was identified. The full length cDNA of clone 4.21 contained 2527 nucleotides encoding 769 amino acids of which 100 are glutamine residues (13%). Antibodies raised against recombinant protein encoded by a partial cDNA sequence (clone 73-k) recognized a 53 and 80 kDa protein in O. volvulus larval and adult parasite extracts, respectively. The antibodies localized the native protein in the cuticle, hypodermis, secretory vesicles and in granules of the glandular esophagus of larvae and in the hypodermis and the cuticle of adult worms. The recombinant 73-k polypeptide (r73) was recognized by 90-100% of sera from PI and infected individuals from Liberia, but only by 67% of similar groups from Ecuador. r73 specific IgG2 and IgG3 levels in the PI from Liberia and Ecuador, respectively, were significantly lower than in the infected, whereas the r73 specific IgG1/IgG3 or IgG1/IgG2 in the PI and the infected individuals from Liberia or Ecuador, respectively, were similar. The IgG4 specific antibody response in the PI from Liberia and Ecuador were lower than in the infected. The T-cell proliferative responses to r73 in infected individuals from Cameroon were found to be inversely correlated with their levels of microfilariae.

Do Filarid Nematodes have a Vascular System

The organization of filarid nematode anatomy has evolved for optimal acquisition of nutrients and delivery of biosynthetic products. The hypodermal cells are key factories for proteins destined for both the hypodermis and the cuticle. To optimize nutrient acquisition, the metabolically active hypodermal cells have cell bodies that bulge as four cords along the length of the worm, putting them in close apposition to the gut epithelium1xWeber, P. Tropenmed. Parasitol. 1984; 35: 221–230PubMedSee all References, 2xStrote, G. and Bonow, I. Parasitol. Res. 1991; 77: 526–535Crossref | PubMed | Scopus (13)See all References.The hypodermis comprises a syncytia, which provides one pathway by which protein synthesized in the hypodermal cell bodies can reach the worm’s surface or cuticle. We propose that a second pathway exists through a network of intercellular channels or conduits connected to the pseudocoelom in the nematodes (Fig. 1Fig. 1). Previously, these structures were referred to as basal lamina, intercellular space or pseudocoelomic membrane1xWeber, P. Tropenmed. Parasitol. 1984; 35: 221–230PubMedSee all References, 2xStrote, G. and Bonow, I. Parasitol. Res. 1991; 77: 526–535Crossref | PubMed | Scopus (13)See all References, 3xChitwood, B.G. and Chitwood, M.B. : 1–50See all References, 4xLevine, N.D. : 344–409See all References.Fig. 1Distribution of Onchocerca proteins in proposed channel network. Aldolase (a): Note grains of immunogold labeling of aldolase in L3, contiguous to cord cell (HC), between muscle cells (M), in cuticle (C), the channel network (arrowheads) and the connections to pseudocoelomic space (arrow). Ov-Alt-1 (b): Note labeling of Ov-Alt-1 in the disintegrating granules (G) of L3, Day 1 in culture, the channels (arrowheads) connecting the esophagus to the cuticle (C), and throughout all layers of the cuticle of L3 by Day 1 in culture. Scale bars = 500 nm.View Large Image | Download PowerPoint SlideThese structures form a network, which may in fact be akin to vascular channels, filled with pseudocoelomic fluid, and capable of efficiently transporting proteins to dispersed regions of the nematode body. Muscular contraction during worm movement could provide the hydrostatic pressure required to circulate fluid through the network. Fig. 1Fig. 1 shows the localization of the Onchocerca volvulus aldolase (encoded by clone Ov-S104, accession no. U96178), a protein synthesized in the hypodermal cell bodies (cords), but then dispersed along the channel network. In a previous study, synthesis of the Onchocerca cysteine protease LOVCP was also traced to hypodermal cells and the glandular esophagus. It, too, appeared to be secreted into the channel network and then transported to the hypodermal–cuticular interface5xLustigman, S. et al. J. Biol. Chem. 1996; 271: 30181–30189Crossref | PubMed | Scopus (82)See all References5. Another protein, Ov-Alt-1, encoded by the cDNA clone Ov-alt-1 [accession no U96176, and homologous to Di 20/22 and Bm-alt-1 (6xFrank, G.R., Tripp, C.A., and Grieve, R.B. Mol. Biochem. Parasitol. 1996; 75: 231–240Crossref | PubMed | Scopus (32)See all References, 7xGregory, W.F., Blaxter, M.L., and Maizels, R.M. Mol. Biochem. Parasitol. 1997; 87: 85–95Crossref | PubMed | Scopus (67)See all References)] was found to be present only in the granules of the glandular esophagus in the vector derived L3 stage (G.T. Joseph, T. Huima and S. Lustigman, unpublished). However, when the larvae initiated the molting process (L3, Day 1 in culture), the protein appeared to be transported from degranulating esophageal cells to the cuticle via channels (Fig. 1bFig. 1b).Electron micrographs of filarids provide only a static vision of the proposed channel network. Proof of its existence must come from further physiologic studies. We encourage investigators working with different parasitic nematodes to evaluate techniques for introducing tracer material that could be followed by dynamic microscopy in living worms. Membrane networks similar to those we have seen in Onchocerca are also found in Wuchereria1xWeber, P. Tropenmed. Parasitol. 1984; 35: 221–230PubMedSee all References1 and Brugia (J.H. McKerrow, T. Huima and S. Lustigman, unpublished).