Identification and characterisation of a novel repetitive antigen from Onchocerca spp.

MOLECULAR AND

ELSEVIER

Molecular and Biochemical Parasitology 63 (1994) 49 57

BIOCHEMICAL PARASITOLOGY

Identification and characterisation of a novel repetitive antigen from Onchocerca spp. Julian Catmull, Dan Zhang, Florence Ruggiero, David B. Copeman, David J. Miller* Department of Chemistry and Biochemistry, James Cook University of North Queensland, Townsville, Queensland 4811, Australia (Received 3 June 1993; accepted 1 October 1993)

Abstract

A novel repetitive antigen from the cattle parasite Onchocerca gibsoni was shown to be recognised by sera from humans infected with Onchocerca volvulus, Wuehereria bancroftii or Brugia malayi. The O. gibsoni protein was produced in a recombinant form, and antibodies raised to this protein used to screen eDNA libraries for O. volvulus. A series of clones were isolated which encoded repetitive regions very similar to those in O. gibsoni, but interspersed between these were longer repeating units which we have not so far found in O. gibsoni. The repetitive antigen was shown to be of high molecular weight and present only in the insoluble (membrane) fraction of O. gibsoni microfilariae. Immunofluoresence techniques demonstrated that the antigen was associated both with muscle and with specific membrane layers, including a peripheral layer which corresponds to either the outer hypodermis or an inner region of the cuticule in adult female O. gibsoni. In many respects, the proteins encoded by the O. gibsoni and O. volvulus eDNA clones resembled repetitive antigens from several distantly related eukaryotic parasites, and a possible common role in immune evasion is discussed.

Key words. Onchocerca gibsoni; Onchocerca volvulus; Microfilariae; eDNA library; Sowda; Repetitive antigens

1. Introduction * Corresponding author. Tel.: 61-77-814473; Fax: 61-77251394; e-mail: [email protected].

Abbreviations." Ag, antigen; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate buffered saline; GST, glutathione S-transferase; HRP, horseradish peroxidase; SDS, sodium dodecyl sulphate; PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin; DAB, diaminobenzidene; FITC, fluoroscein isothiocyanate. Note: Nucleotide sequence data reported in this paper have been submitted to the GenBank T M data base with the accession numbers U01040 (OVM1), U01050 (OG11), U01302(OVGI), U01315 (OVKI), U01316 (OVG3usp) and U01324 (OVG3rsp). 0166-6851/94/$7.00 ~) 1994 Elsevier Science B.V. All rights reserved SSDI 0 1 6 6 - 6 8 5 1 ( 9 3 ) E 0 1 7 1 - 4

The parasitic n e m a t o d e Onchocerca gibsoni c o m m o n l y infects cattle in northern Australia and, in its natural host, is the best available animal model for the h u m a n pathogen, O. volvulus. The microfilariae o f O. volvulus, released in large n u m b e r s by adult female worms, are responsible for the pathological s y m p t o m s associated with 'river blindness'. One m a j o r advantage o f O. gibsoni is that in this case the availability o f material has facilitated the construction o f stage-specific e D N A libraries, which has not been possible for

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J. Catmull et al./Molecular and Biochemical Parasitology 63 (1994) 49-57

other Onchocerca spp. We recently reported the construction of a cDNA library for microfilariae of O. gibsoni, and the nucleotide sequence of a cDNA from this library which encoded a repetitive antigen recognised by sera from human individuals infected with O. volvulus [1]. c D N A clone pOG#11 encoded a protein consisting of repeating units of 29 amino acids [1]. Below we report characterisation of the corresponding O. gibsoni cellular antigen, and nucleotide sequences of clones isolated from O. volvulus cDNA libraries using antibodies raised to the protein encoded by pOG#11.

2. Methods

2.1. Human serum samples. Aliquots of human serum from single individuals infected with Onchocerca volvulus, Brugia malayi or Wuchereria bancroftii were kindly supplied by N. Weiss at the W H O serum bank. Small volumes of pooled serum from individuals carrying the sowda form of onchocerciasis were also supplied by the same source. The sowda trait is defined as clinical onchocerciasis in the absence of microfilaraemia. 2.2. Production of recombinant protein and antibodies. A Bsu36I/EcoRI fragment corresponding to nt 34-435 of the pUC18 clone pOG#11 [1] was subcloned into the expression vector pGEX-2T [2]. The resulting glutathione S-transferase (GST)-fusion protein was purified on glutathione agarose, cleaved with thrombin on the (glutathione) affinity column, and thrombin removed from the eluate by chromatography on heparin Sepharose. Polyclonal antibodies to the recombinant protein were raised in rabbits (New Zealand white) essentially as described by Mayer and Walker [3]. 2.3. Antibody capture enzyme-linked immunosorbent assay. The ELISA protocol used was based on Voller and Bidwell [4], and Langone and Van Vunakis [5]. All incubations were carried out at room temperature (22-24°C). Between each step the ELISA plates were rinsed 4 times with PBS (phosphate-buffered saline: 0.14 M NaCI/ 2.7

mM KC1/ 10 mM Na2HPO4/ 1.8 mM KH2PO4/ pH 6.8-7.2) containing 0.05% (v/v) Tween 20. Nunc maxisorb ELISA plates were coated for 2 h with 50 ng of recombinant protein in 100 #1 of 15 mM Na2CO3/35 mM NaHCO3, pH 9.6, followed by blocking for 1 h using 200 /~1 of 0.4% (w/v) casein plus 0.05% (v/v) Tween 20 in PBS per well. After washing, primary antibody diluted 200-fold (in 100/~1 blocking solution) was added. After 1 h the wells were washed. The secondary antibody (horseradish peroxidase (HRP)-conjugated rabbit anti-human lgG; Dako P-214) was added at a 1:500 dilution (in 100/~1 blocking solution) and after a 1-h incubation was removed. After a final rinse, 0.5 mg.ml-I 2,2'-azino-di-(3ethylbenzthiazoline sulfonate) plus 0.0075% (v/v) H202 in 200 #1 peroxidase substrate buffer (0.03 M citrate containing 0.04 M Na2HPO4, pH 4.2) was added to each well and following a 1-h incubation the absorbance was read at 405 nm.

2.4. Western blotting. Western blotting was conducted essentially as described by Towbin et al. [6], after Laemmli-type [7] sodium dodecyl sulphate polyacrylamide gelelectrophoresis (SDSPAGE). Strips of western blots were probed under the conditions described for ELISA (above), except that diaminobenzidene (DAB) was substituted as substrate (see screening of cDNA libraries below). Western blots were continually rocked during processing, and incubations were carried out in 2 ml volumes of buffers. 2.5. Immunofluorescence techniques. Parasite material was embedded in OCT resin (Tissue-Teck) and allowed to freeze on a liquid-nitrogen cooled surface. Cryostat sections (5-7 /~m) were transferred to 1% gelatin coated microscope slides and fixed in cold acetone for 10 min. Sections were blocked using either 1% (w/v) BSA or 0.4% (w/v) casein in PBS for 1 h (room temperature), and then incubated for 1 h with primary antibody. Sections were then washed (3 × 5 min) using a gentle stream of PBS, before incubating for 1 h with fluoroscein isothiocynate (FITC)-conjugated secondary antibody. After washing (PBS; 3 × 5 min), slides were mounted with a drop of PBS:glycerol (1:1), the coverslips sealed with nail

51

J. Catmull et al./Molecular and Biochemical Parasitology 63 (1994) 4 ~ 5 7

polish, and sections were examined on a Leitz epifluorescence microscope (filter 2). Fluorescence was recorded on Ektachrome 400 film. Experimental controls included (a) omission of primary antibody, and (b) use of pre-immune serum.

2.6. Screening cDNA libraries. 2gtl 1 c D N A libraries were screened using antibodies as described by Huynh et al. [8]. Nitrocellulose membranes were blocked using PBS containing 5% (w/v) skimmed milk powder and 0.1% (v/v) Nonidet P-40 for 1 h at room temperature. All subsequent steps were at 4°C. Membranes were incubated for 12-16 h with primary antibody (1:1000; diluted in blocking solution), washed (5 × 5 min in PBS containing 0.1% (v/v) Nonidet P-40) and then incubated for 3~ , h with peroxidase-conjugated secondary antibody (HRP conjugated swine anti-rabbit IgG; DAKO-P217; 1:1500 diluted in blocking solution). After washing, the membranes were incubated with 0.6 mg m1-1 DAB and 0.018% (v/v) H202 in substrate buffer (0.03 M citrate containing 0.04 M Na2HPO4, pH 4.2) at room temperature. After plaque purification, O. volvulus inserts in cDNA clones were subcloned into either pUC18 or p G E M l l . Plasmid templates were used for D N A sequencing using the chain termination method [9,10].

3. Results 3.1. Reaction of human sera with recombinant O. gibsoni protein. In antibody-capture ELISA experiments, using plates coated with 50 ng of recombinant protein per well, pooled serum from patients with 'sowda' onchocerciasis (25 individuals per pool; Liberian and Yemen 'sowda' onchocerciasis) reacted at dilutions down to 1:3200, whereas control sera did not react significantly at 1:50. Fig. 1 summarises antibody-capture ELISA results using serum samples from single individuals infected with a range of filarial parasites, and implies a high degree of serum cross-reactivity to the O. gibsoni protein. Wide and similar ranges of serum reactivity were observed for individuals in-

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Group Fig. I. Apparent serum cross-reactivity to the recombinant protein. Serum samples from single individuals infected with a range of filarial parasites were subjected to antibody capture ELISA, using a fixed primary antibody dilution of 1:200. The results are grouped as follows: C, controls (n = 22); Ov, O. volvulus (general onchocerciasis; n - 6 5 ) ; Bm, Brugia malayi ( n - 20); Wb, Wuchereria bancrofti ( n - 92). The points represent the mean values, and the bars give the standard deviation.

fected with O. volvulus ('general' onchocerciasis), Wuchereria bancrofti and Brugia malayi. However, the reactions given by the pooled sowda sera were higher than those given by individuals with general onchocerciasis; under the standard antibody-capture conditions, the ELISA values given by the Liberian and Yemen 'sowda' pools were 2.4 and 3.1 respectively. Antigenic cross-reactivity of this type may reflect functional conservation of some nematodespecific component between these filarial parasites or, alternatively, it may be a consequence of conservation of a component required for interaction with the host immune system. The similarity between the Onchocerca proteins and repetitive antigens from Plasmodium falciparum (see below) supports the latter of these explanations.

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J. Catmull et al./Molecular and Biochemical Parasitology 63 (1994) 49--57

3.2. Characterisation of the O. gibsoni cellular antigen. Western blotting experiments demonstrated that the antigen encoded by pOG#11 was of high molecular weight and was associated with insoluble (membrane) fractions of O. gibsoni microfilariae. When antibodies to the recombinant protein were used to probe western blots, a single protein of high ( > > 340 kDa) molecular weight was detected (Fig. 2). This antigen was associated exclusively with insoluble fractions; no reaction was observed when western blots or soluble extracts (prepared by grinding microfilariae in PBS under insoluble fraction A B !

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Fig. 2. Distribution and apparent molecular weight of the cellular antigen encoded by pOG 11. The figure shows strips of western blots (7% acrylamide gels) loaded with either 30 gg of the insoluble protein fraction or 20/~g of soluble protein fraction prepared from uterine microfilariae of O. gibsoni. Pairs of strips were probed with either (A) rabbit antiserum to the recombinant protein or (B) pre-immune serum. Primary and secondary antibody were used at 1:200 and hl000, respectively. The bars at the left of the figure indicate the positions of migration of molecular weight standards on the same gels, and the arrow indicates the top of the resolving gel in each case. Note that at higher acrylamide concentrations, the antigen did not migrate into the resolving gel.

liquid nitrogen followed by centrifugation at 12000 x g for 10 min) were probed with the antibody to the recombinant protein (Fig. 2). When antibody to the recombinant protein was also used to probe sections of whole O. gibsoni nodules and isolated adult worms in immunofluorescence experiments (Fig. 3), the patterns of labelling observed were always highly specific; preimmune serum controls were uniformly blank and bovine tissue, which served as an internal control in many experiments, was likewise always unlabelled. As well as both male and female adult worms, early embryos and microfilariae in utero each displayed specific immunofluorescence labelling patterns (Fig. 3). In general the nematode muscle tissues showed the most intense labelling. In transverse sections, the dorsal and ventral muscle bundles of the adult (Fig. 3a and b) worms were heavily labelled, as well as uterine muscle of the female. Larvae cross sectioned in utero displayed a characteristic labelling pattern (Fig. 3c), corresponding to the dorsal and ventral muscle bundles. In longitudinal section the larvae appeared to be labelled peripherally over their full length (Fig. 3d). In addition to immunofluorescence associated with muscle, several distinct membrane layers were specifically labelled in sections. In the adult female worm, strong immunofluorescence was associated with 2 distinct membrane layers: (1) a peripheral membrane, corresponding to either the outer hypodermis or an inner cuticular layer, and (2) an inner uterine membrane (Fig. 3a). Fig. 3e shows transverse sections of an adult female O. gibsoni with early embryonic stages in utero; here the pattern of fluorescence observed from the embryos was associated with the cell wall of every cell, with none of the muscle-specific labelling characteristic of other stages being apparent. Although we believe that antigen plays an important role in interaction with the host immune sytem, expression from early embryogenesis through to adulthood suggests that the protein may have other essential function(s), such as structural or cell attachment roles. The diversity of tissues showing labelling is also suggestive of multiple functions. Despite the strong immunofluorescence results,

J. Catmull et al./Molecular and Biochemical Parasitology 63 (1994) 49-57

attempts to use the antibody at the electron microscope level were uniformly unsuccessful. Post-em-

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Fig. 3. Immunolocalisation of the cellular antigen encoded by pOG 11. The figs. show the pattern of fluorescence after probing sections of various stages o f O. gibsoni using antibody (1:500) to the recombinant protein and then detecting bound primary antibody using FITC-conjugated secondary antibody (1:30). (A) adult female worm in cross section; (B) adult male worm in cross section; (C) uterine microfilariae in cross section; (D) longitudinal section of adult female worm, with microfilariae in utero; (E) e m b r y o s (blastula stage) sectioned in utero; (F) controis: cross sections o f adult female worm were probed as above, using (i) pre-immune serum (1:100 dilution) or (ii) secondary antibody only. The positions of the outer hypodermis (oh), hypodermis (h), uterine muscle bundles (umb), inner uterine m e m b r a n e (ium), uterine microfilariae (umf), dorsal (dm) and ventral (vm) somatic muscles (sm), gut (g), eggs and embryos (ee) are indicated as appropriate. The scale bars represent 50 #m.

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J. Catmull et al./Molecular and Biochemical Parasitology 63 (1994) 49 57

hyde or 4% paraformaldehyde (in PBS), after which the materials were embedded in either LRgold or Lowicryl-K4 and sectioned conventionally. In all cases, antigenicity was completely lost.

quence encoded by the O. gibsoni clone pOG#11. In the O. volvulus clones OVG1 and OVM1, the pattern of repeating units was A A B A A A B A A (where each A denotes an O. gibsoni-like 29-amino acid repeat unit and each B denotes a 41-amino acid O. volvulus repeat unit), and although OVK1 was shorter, containing only 6 repeat units, the pattern of repeats was the same (AABAAA). The sequence data of OVG3 are incomplete, however, the pattern of repeats is the same as in the other clones. Thus, the periodicity of the O. volvulus repeats varies from 99 amino acids (AAB) to 128 amino acids (AAAB). The 29-amino acid repeats in the O. volvulus clones were very similar to those of O. gibsoni; the only consistent difference was substitution of an aspartic acid or lysine residue at position 23 in the O. volvulus repeats for a glutamic acid residue in the O. gibsoni sequences. The observed high degree of sequence conservation extended also to

3.3. Characterisation of cDNA clones from O. volvulus. A series of clones were isolated by screening O. volvulus c D N A libraries (kindly supplied by J. Donelson) using antibodies raised against the recombinant O. gibsoni protein. Four c D N A libraries were screened and, from a total of approximately 24400 plaque-forming units, 9 strongly reacting clones were isolated. Each of the O. volvulus clones was found to encode regions closely resembling the O. gibsoni 29-amino acid repeat units, but interspersed between these regions were repeat units of 41 amino acids, which differed substantially to the O. gibsoni repeats. In figure 4 the deduced amino acid sequences encoded by 4 of the O. volvulus clones are shown aligned with the se(A) IM)O#11 O%'111 OVGI DVO3(USP) OVG3(RSp} (YCZI pOG#ll 0%'O1 OVG3(USP) OVG3{RSP) OVZl

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................. A E V K M E Q P K E E I ~ E ~ 4 K P K E K V V D D K K K K A E V K M E Q ~ K E E I V E E M K ~ K E K V V K D K K E K A V E N L E K S E I M N F A K D I E H E F V D C V I L S S T ................. A E V K M E Q • K E E I v E E M K P K E K V V D D K K K K A E V K M E Q P K E E I V E E M K P K E K V V K D K K E K A V E N L E K S E I M N 8 A K D I E H E F V D V C I L S S T ................. A E V K M E Q P K E E I v E ~ 4 K p K E K v V D D K K K K A E V K M E Q P K E E I V E E M K P K E K V V K D K K E K A v E N L E K S E I M N F A K D I E H E F V D V C I L S S T ............. V R ~ K E V K M E Q P K E E I V E ~ 4 K ~ K E K v V D D K K K K A E V K M E Q P K E E I V E E M K P K E K V V K D K K E K A V E N L E K S E I M N F A K D I E H E F V D V C I L S S T ................. A E V K M E Q • K E E I V E E M K P K E K v v A D K K K K A E V K M E Q P K E E I V E E M K • K E K V V K D K K E K A V E N L E K S E I M N F A K D I E H E F V D V C I L S S T

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Fig. 4. (A) Alignment of the repetitive amino acid sequences encoded by the c D N A s from O. gibsoni and O. volvulus. Bars above the sequences indicate the positions of the 29-amino acid repeat units. Non-conservative substitutions (including differences to the consensus) are shown in bold. (B) Schematic representation of the pattern of repeat units encoded by the c D N A clones. The 29- and 41-amino acid units are designated as A and B, respectively. Note that for clone OVG3, the complete nucleotide sequence has not been determined, however, the number of repeats and their order are known to be as shown.

J. Catmull et al./Molecular and Biochemical Parasitology 63 (1994) 49-57

the nucleotide level (data not shown), implying that codon useage is strongly constrained. As in a number of other parasite antigens (see discussion), the repetitive protein encoded by the O. gibsoni e D N A clones is predicted to be highly

55

hydrophilic, and to contain predominantly alpha helices (Fig. 5a). Whilst these features also apply in general to the O. volvulus sequences, each of the 41-amino acid units contains a hydrophobic region (residues 20-30) and clustered serine residues are predicted to result in a short coiled section at residues 27 31 (Fig. 5b).

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The O. gibsoni e D N A clone encodes a repetitive region of a high molecular weight protein which is associated with membranes and insoluble fractions in all stages of the parasite. Whilst the western blotting experiments demonstrated that the antigen encoded by the O. gibsoni eDNA clones was associated with membranes (Fig. 2), the predicted amino acid composition is consistent with a hydrophilic protein and the recombinant protein was soluble. These results imply that the eDNA clones which we have isolated encode hydrophilic domains of a membrane protein. There are precedents for this; for example, the 8-amino acid repetitive region of the Plasmodium falciparum antigen RESA [11] and the 3- and 6-amino acid repeat regions of the Pfl 1-1 antigen [12,13] are hydrophilic domains in membrane-associated parasite proteins. In immunofluorescence experiments antibody to the recombinant O. gibsoni protein recognised 2 distinct regions; the muscle and a layer under the cuticle (Fig. 3). The strong fluorescence associated with muscle initially suggested that the antibody may have recognised an abundant muscle structural protein such as myosin. However, the western blotting experiments demonstrated that the antigen was of higher molecular weight than any previously-characterised nematode muscle structural protein. Although the proteins encoded by these c D N A clones had statistically significant matches to alpha-helical regions of a number of structural proteins, these were attributed to similar amino acid biases rather than to sequence similarities. In many respects, the proteins encoded by these O. gibsoni and O. volvulus cDNA clones resembled repetitive antigens from several distantly related

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J. Catmull et al./ Molecular and Biochemical Parasitology 63 (1994) 4 9 3 7

eukaryotic parasites. For example, the amino acid composition of the O. gibsoni and O. volvulus cDNA-encoded repetitive proteins was strongly biased and similar to those of several of the immunodominant proteins from Plasmodium falciparum, including RESA [11], Pfll-1 [12,13] and G L U R P A [14]. Like O G l l and OVX (X being representative of all related O. volvulus clones isolated here), the repetitive regions of each of these proteins are glutamic acid-rich (48% of the RESA 8-amino acid repeat region; 32.5% of the GLURPA 19-amino acid repeat region and 41% of the Pfll-1 9-amino acid repeat region, compared with 27% of the OG11 protein), and have valine and lysine as major components. Like each of these regions, the O G l l and OVX repetitive regions are predicted to consist predominantly of alpha helices, and to be hydrophilic (the exception is the 9-amino acid repeat region of Pfl 1-1, for which alternating hydrophobic and hydrophilic sections are predicted). The similarity between these antigens from Onchocerca and P. falciparum is likely to reflect functional constraints rather than true homology (see below). In terms of amino acid composition, the G L U R P A repeat region had the most overall similarity to O G l l and OVX; G L U R P A contains 14 tandem repeats of a 19-amino acid unit [14]. There is, however, a higher degree of sequence similarity between the 29-amino acid units and the 9-amino acid repeat region of Pfl 1-1 at 6 positions (of 9) and a further 2 positions are conservative substitutions (Fig. 6). One view of the role of repetitive regions in parasite antigens is that they may present a battery of cross-reactive epitopes to the host, which interfere with the affinity maturation of poten-

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Fig. 6. Alignment of amino acid residues 9 17 of the hypothetical O. gibsoni polypeptide with the R9 repeat unit encoded by the 3' region of the P. falciparum l l I gene [12] and the 8- (2 copies) and 4-amino acid repeats encoded by the 3' end of the P. falciparum RESA gene [11].

tially protective responses [15]. Schofield [16] has refined this hypothesis, suggesting that such repetitive regions may function in immune evasion by inducing T-independent B-cell activation. Well characterised T-independent antigens, such as dextrans, pneumococcal polysaccharide and ficoll, consist of multiple repeat units and present identical antigenic epitopes; cross-linking of membrane immunoglobulins on the surface of B-cells by such tandemly-repeated epitopes provides a T-independent activation signal [17]. Schofield [16] proposes that parasite repetitive protein domains similarly act as tandemly repeated epitopes and thus initiate T-independent responses. Further, the repetitive domains may cause epitopic suppression; as perfect or designer B-cell epitopes, they may suppress antibody responses to adjacent regions of the protein. Many of the characteristics of repetitive parasite antigens, including those which we report here, are consistent with the Schofield hypothesis. For example, the strongly biased and similar amino acid composition, and the extensive serological cross-reactivity observed between repetitive proteins from distantly related parasites are consequences of the constraints applying for construction of efficient B-cell epitopes; amino acid usage and overall 3-dimensional structure must be highly constrained, inevitably resulting in cross-reactivity. As figure 1 shows, serum from patients with a variety of filarial infections reacted with the recombinant O. gibsoni protein, and indirect evidence implies that the recombinant O. gibsoni may present a single repetitive epitope when used as an antigen. For example, despite generating high titres in experimental animals (the titre of the rabbit serum used in immunofluorescence was 1:25 000-1:50 000, using the antibody-capture ELISA protocol given above), immunoprecipitation only occurred under conditions of vast excess of antibody (10 pl of undiluted serum was required to immunoprecipitate < <0.25 pg of OGI1 protein). The loss of antigenicity, leading to our inability to conduct localisation experiments at the electron microscope level, is also understandable if a single epitope is immunodominant. Because epitopic immunosuppression is c/s-act-

J. Catmull et al./Molecular and Biochemical Parasitology 63 (1994) 49-57 ing ( w h e r e as the p r o t e c t i v e effect w h i c h A n d e r s [15] p r o p o s e s is trans-acting), it is i m p o r t a n t to c h a r a c t e r i s e the n o n - r e p e t i t i v e d o m a i n s o f the O G l l c e l l u l a r a n t i g e n . A s the r e p e t i t i v e r e g i o n s a p p e a r to be e x t e n s i v e (a 1400 b p O. volvulus cDNA clone contained only repetitive motifs), and internal priming of cDNA synthesis apparently o c c u r s r e l a t i v e l y f r e q u e n t l y ( d u e to r u n s o f p o l y ( A ) in t h e r e p e a t m o t i f s ) , i s o l a t i o n o f c D N A c l o n e s e n c o d i n g n o n - r e p e t i t i v e d o m a i n s is likely to be difficult.

5. Acknowledgments W e t h a n k D r . N. W e i s s at the W H O for s u p p l i n g the h u m a n sera a n d D r . J. D o n e l s o n f o r s u p p l y i n g t h e O. volvulus c D N A libararies. T h e t e c h n i c a l a s s i s t a n c e o f M . F l a n a g a n a n d L. R e i l l y is g r a t e f u l l y a c k n o w l e d g e d .

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