INTRODUCTION

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Onchocerca volvulus is the causative agent of onchocerciasis, a disease that affects about 20 million people in Africa, the Arabian Peninsula, and Central and South America. The control of this disease is largely dependent on administration of annual doses of ivermectin, a drug that is able to kill the microfilariae but not the adult worms (22).

The spectrum of O. volvulus infection varies from asymptomatic microfilardermia often associated with immunological hyporesponsiveness to severe skin and eye diseases including onchodermatitis and blindness. Establishment of infection and disease development are dependent on the specific antigen recognition and immune response of the host. The complex immune response is triggered by numerous different filarial antigens at the same time. To elucidate the mechanism of the immune response, it is essential to explore the specific reactions to individual, native parasite antigens.

The glutathione S-transferases (GSTs) are a family of detoxification enzymes that catalyze the conjugation of reduced glutathione (GSH) to xenobiotic and endogenous electrophilic compounds (25, 49, 55, 56). Besides having a role in detoxification, they are involved in the protection of tissues against oxidative damage and in the intracellular transport of hydrophobic substrates as noncatalytic carrier proteins (ligandins) (for recent reviews see references 14, 26, and 52). In a parasitic context it is especially important to consider their function in the regulation of the stress response, in the detoxification of lipid peroxidation products, in drug resistance, and possibly in the modulation of the host immune defense mechanisms (8, 16, 42).

Several studies demonstrate the participation of filarial GSTs in the defense against oxidative stress (34, 35). The potential to protect the parasite against host immunity makes them attractive candidate vaccine antigens (11). The significant differences between the tertiary structure of the helminth GSTs and that of the host enzymes make the GSTs promising chemotherapeutic targets (9).

From the perspective of antibody reactivity, the most immunogenic filarial antigens that have been identified include paramyosin, tropomyosin, OvALT-1, SXP1, and chitinase (1, 18, 21, 30). The ease of recombinant production of these and other potential vaccine molecules has made vaccination trials using various animal models possible. However, as a result of this, the role that carbohydrates and other nonprotein determinants play in the immune responses to parasites has been undervalued. However, it is well recognized that most of the immunodominant antigens either are excreted or secreted proteins or are abundant constituents of the nematode surface that are glycosylated (36, 38).

The O. volvulus GST1a and -1b (OvGST1a and -1b) are unique GSTs in that they are glycoproteins possessing signal peptides that are cleaved off in the process of producing the mature protein. The mature protein starts with a 25-amino-acid extension not present in other GSTs. The ultrastructural localization of the secretory OvGST1a and -1b in parts of the cuticle and in the outer lamellae of the hypodermis is consistent with the fact that they are secretory proteins (31). The structures of the N-glycans have been determined, and a three-dimensional model has been created to demonstrate their localization profile. By virtue of the potential significance of OvGST1a and 1b as immunoprophylactic targets, we have concentrated our efforts on the immunological relevance of the N-glycans and of the uncommon N-terminal extension.

(This work was conducted by A. Sommer in partial fulfillment of the requirements for a PhD from the University of Hamburg, Hamburg, Germany.)

	MATERIALS AND METHODS

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Parasites, preparation, and purification of native OvGST1a and -1b. Nodules containing O. volvulus female adults were removed from untreated patients with generalized onchocerciasis in Benin, as described previously (2). Nodulectomies for research purposes were approved by the Ethics Commission of the Medical Board Hamburg. Adult worms were homogenized on ice with a glass and glass potter in phosphate-buffered saline (PBS), pH 7.4, containing 0.1 mM phenylmethylsulfonyl fluoride. The homogenate was centrifuged for 1 h at 100,000 × g, and the native OvGSTs were purified from the supernatant as previously described (32).

Production of recombinant OvGST1a and -1b. The different OvGST constructs were cloned into expression vector pJC20 and transformed in Escherichia coli strain BL21. Expression of recombinant OvGST1a (rOvGST1a) and -1b, followed by purification using GSH-Sepharose, was done as previously described (33).

Site-directed mutagenesis. To obtain the mutation of M25 to A25 in OvGST1a, PCR was performed with the specific sense oligonucleotide 5'-GCTACAACCTCAAGCGGAAAAGTACACATTAAC-3' and antisense oligonucleotide 5'-GTTAATGTGTACTTTTCCGCTTGAGGTTGTAGC-3' using 1:100 (vol/vol) OvGST1a/pJC40 as the template. Following PCR using Pfu polymerase, the products were incubated with DpnI for 1 h at 37°C and 1/10 (vol/vol) was transformed in E. coli strain DH5.

Synthesis of two 17-mer peptides of the N-terminal extension. The following overlapping peptides of the N-terminal extension, coupled to poly-L-lysine, were synthesized at IPF PharmaCeuticals GmbH: (ASSNANQAITSENSIKP)₈K₇A and (AITSENSIKPKGKLQPQ)₈K₇A.

Modeling, model refinement, and structure validation. Three-dimensional models were generated based on the crystal structure of the squid sigma class GST (PDB code: 1GSQ) (19). The primary amino acid sequences of OvGST1a and the squid GST have a similarity of 45%, with 25% identical amino acids. The modeling of the three-dimensional structure of OvGST1 followed a standard stepwise procedure, starting with an alignment of the target sequence onto the template structure using the Malign program. Due to the missing N-terminal 25 amino acids in the template, this stretch of amino acids was omitted in the modeling steps. The initial model was generated by the MODELLER4 package (48), and a first energy minimization step was performed using the CHARMM force field (7). Following the superpositioning of the protein structures, refinements were carried out with the AMBER force field (45). Subsequently, the energy minimization was performed with 100 steps of steepest descent followed by 100 steps of a quasi-Newton minimization (46) to alleviate steric clashes between atoms and obtain a rational peptide geometry. The quality of the model was assessed using different validation tools. The OvGST1a structure was validated with the program PROCHECK (29) at each step of the model building. No significant geometric violations were detected in the final model. Molecular visualization was carried out with the programs MOLSCRIPT and Raster3D (41).

Deglycosylation of the native OvGST1a and -1b. The native OvGSTs were dialyzed against 10 mM EDTA-50 mM sodium phosphate, pH 7.5, heated at 80°C for 2 min, and then incubated overnight with the appropriate amount of N-glycosidase F at 37°C.

ELISA. The reactivity of the N-glycans to patient sera was assessed by an enzyme-linked immunosorbent assay (ELISA). The optimal coating concentration of the recombinant and native antigens and serum dilutions were determined by a checkerboard titration ELISA using a pool of sera from O. volvulus-infected individuals. Following dialysis against 0.1 M sodium carbonate buffer, pH 9.6, the glycosylated and deglycosylated native OvGSTs were covalently coupled to activated CovaLink NH plates (Nunc) at a concentration of 1 µg/ml. The rOvGSTs were used at a final concentration of 2 µg/ml. The antigens were incubated overnight at room temperature, washed three times with distilled water, and then blocked with 5% (wt/vol) dry milk-PBS, pH 7.5. All sera were used at a dilution of 1/400 (vol/vol). Bound antibodies were detected with peroxidase-conjugated antibodies against human immunoglobulin G (IgG) using tetramethylbenzidine as the substrate. Between each incubation step, five washing cycles with PBS-16.9 g of NaCl/liter-10 g of MgCl₂/liter-0.05% Tween 20 were performed. After the substrate reaction was stopped by 0.5 M H₂SO₄, the optical density at 450 nm (OD₄₅₀) was measured. Sera from confirmed microfilaria-positive patients with generalized onchocerciasis (38 samples) were obtained from Benin and Guinea (5, 6). Negative controls included experiments using irrelevant proteins for coating and experiments performed without coating or serum. To exclude the influence that preheating of OvGST1a and -1b before deglycosylation has on antibody recognition, comparative ELISA of the native proteins was performed with and without preheating and overnight incubation at 37°C. African control sera (four samples) and sera from Europeans not exposed to filariae (five samples) were used to determine the cutoff level. As a positive control, one test serum of a patient with generalized onchocerciasis with known high antibody concentration was included on each test plate, and only intra- and interassay variations less than 10% were accepted.

Statistical analysis. Differences between the data in two groups were analyzed by the Mann-Whitney U-test and the paired and unpaired Wilcoxon test. A P value of <0.05 was regarded as significant.

Preparation of native OvGST1a and -1b for mass spectrometry (MS) analysis. Purified native OvGST1a and -1b were dialyzed against 10 mM Tris-HCl, pH 8.1, followed by heating at 80°C for 2 min. The proteins were incubated overnight at 37°C with trypsin at a 30:1 ratio in 100 mM Tris-HCl, pH 8.1-0.1 mM dithiothreitol-0.02% (wt/vol) NaN₃. Following a second dialysis against 10 mM ammonium hydrogencarbonate, pH 7.4, the peptide mixture was lyophilized and stored at 20°C until use.

HPLC and ESI-MS/MS. The tryptic peptide mixtures (approximately 100 pmol) were analyzed on an Applied Biosystems 172Å microbore high-pressure liquid chromatography (HPLC) system using an Aquapore OD-300 C₁₈ column (1.0 by 100 mm) at a flow rate of 40 µl/min and a linear gradient from 4 to 56% acetonitrile in 0.1% formic acid and 4 mM ammonium acetate. Elution of peptides was monitored by UV absorption at 214 nm and by MS on a TSQ 700 triple-quadrupole instrument equipped with a Finnigan ES ion source connected on line to the HPLC system. In a preparative run, (glyco)peptide fractions were collected manually and subjected to Edman sequencing (Applied Biosystems; model 475A). Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) MS and electrospray ionization tandem mass spectrometry (ESI-MS/MS) analysis are described below.

MALDI-TOF MS. For analysis by MALDI-TOF MS the (glyco)peptide fractions were measured using a matrix of 19 mg of -cyano-4-hydroxycinnamic acid in 400 µl of acetonitrile and 600 µl of 0.1% (vol/vol) trifluoroacetic acid in water. The sample solution was mixed with the same volume of the respective matrix. The concentration of the analyte mixture was 1 to 10 pmol/µl. Determination of the molecular masses was carried out by positive-ion MALDI-TOF MS using a Bruker REFLEX time-of-flight instrument as described by Grabenhorst et al. (15).

ESI-MS/MS. For analysis by ESI-MS/MS the (glyco)peptide samples (approximately 3 µl) were used to fill gold-coated nanospray glass capillaries (Protana, Odense, Denmark). The tip of the capillary was placed orthogonally in front of the entrance hole of a quadrupole time-of-flight mass spectrometer (Micromass, Manchester, United Kingdom) equipped with a nanospray ion source, and a voltage of approximately 1,000 V was applied. For collision-induced dissociation experiments, parent ions were selectively transmitted from the quadrupole mass analyzer into the collision cell. Argon was used as the collision gas, and the kinetic energy was set at around 35 eV. The resulting daughter ions were then separated by an orthogonal time-of-flight mass analyzer (50).

	RESULTS AND DISCUSSION

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Comparison of the amino acid sequences of OvGST1a and OvGST1b. The purification of glutathione-binding proteins from O. volvulus extract, produced by centrifugation at 100,000 × g, by GSH affinity chromatography yielded three proteins of roughly 34, 31, and 26 kDa, representing OvGST1b, OvGST1a, and OvGST2, respectively (31, 32). The average content of purified OvGST1a and -1b was 0.5 µg/adult female worm. Only the fractions containing native OvGST1a and -1b were used for further experiments. Figure 1, lane A, demonstrates the purification of native OvGST1a and -1b. Due to the microheterogeneity of the N-glycans, a broader band between 31 and 33 kDa, representing OvGST1a, is seen whereas OvGST1b is seen as the upper, very faint band. Immunoblot analysis using O. volvulus extract and anti-rOvGST1 demonstrates the same staining pattern (31). This strongly indicates that the observed difference in the quantities of OvGST1a and -1b is not due to the purification procedures, i.e., different affinities for binding to GSH-Sepharose, but rather reflects the situation in the worm.

View larger version (104K): [in this window] [in a new window]   FIG. 1.   Purification and deglycosylation of native OvGST1a and -1b. The proteins were separated on an SDS-12.5% PAGE gel. Lane A, OvGST1a and OvGST1b at 31 to 33 and about 34 kDa, respectively. The relatively broad bands can be explained by N-glycan microheterogeneity. For comparison, rOvGST1a raised in E. coli was also applied (lane B). Lane C, completely deglycosylated proteins after N-glycosidase F treatment; lane D, digestion with endoglycosidase H, leaving a single GlcNAc residue at each glycosylation site in place. Left, migration positions of molecular mass standard proteins.

View larger version (47K): [in this window] [in a new window]   FIG. 2.   Alignment of the amino acid sequences of OvGST1a and OvGST1b. The signal peptide, comprising 25 amino acids, is indicated. Arrows, cleavage site, N-terminal extension, and tryptic cleavage sites. The numbering of the respective amino acids begins at position 1 (in boldface), which corresponds to the signal peptide cleavage site of the mature protein. The numbers above the sequences correspond to the tryptic fragments shown in Table 1. The differences between OvGST1a and -1b are in boldface.

Isolation and characterization of N-linked oligosaccharides. As previously shown (31), the native enzymes are extensively glycosylated. The glycosylation accounts for approximately 13 and 20% (OvGST1a and OvGST1b, respectively) of their apparent masses (Fig. 1, lane A). For comparison the nonglycosylated protein expressed in E. coli (Fig. 1, lane B) as well as the fully deglycosylated peptide backbone after N-glycanase F treatment (Fig. 1, lane C) were run on the same SDS-PAGE gel. To characterize the glycan structure of the purified native proteins, OvGST1a and -1b were subjected to tryptic digestion. There were 27 tryptic cleavage sites for both OvGSTs, with one specific cleavage site for each protein. Cleavage between amino acids 3 and 4 in OvGST1b resulted in specific fragments 1a and 1b, and cleavage between amino acids 69 and 70 of OvGST1a generated specific fragments 10a and 10b (Fig. 2; Table 1). The resulting fragments were separated by microbore HPLC, and the elution was monitored by UV absorption and MS. Figure 3 shows the corresponding UV trace at 214 nm. The peaks identified by HPLC-MS analysis are marked (Fig. 3; Table 1); further peaks correspond to nonpeptide contaminants as well as to trypsin fragments. Most of the nonglycosylated tryptic peptides were identified; peptides <400 Da, however, were not detected in the HPLC-MS analysis (Fig. 3 and Table 1). All OvGST1a peptides bearing a glycosylation site were identified by the characteristic pattern of molecular ions due to the attachment of different glycans (as shown for glycopeptides 1 and 4 in Fig. 4). On account of the small quantity of OvGST1b and the minute differences between both proteins observed, only two specific glycopeptides of OvGST1b were identified. Glycopeptide 1, consisting of amino acids 4 to 18 (GP1_4-18) of OvGST1b, could not be detected and analyzed (Table 1). On all four glycosylation sites of OvGST1a, molecular ions with mass increments of 162 Da were detected, suggesting the presence of high-mannose type structures differing in the number of mannose residues attached, as shown in Fig. 4 for GP1_44-57 and GP4_144-156. The assignment and the ratios of the glycan structures found at individual glycosylation sites of OvGST1a are as follows: for GP1_44-57, Man₅/Man₄/Man₃ ratio = 1:0.32:0.1; for GP2_70-98, Man₅/Man₄/Man₃ ratio = 1:0.51:0.07; for GP3_134-137, Man₅/Man₄/Man₃ ratio = 1:0.13:0.07; for GP4_144-156, Man₅/Man₆/Man₇/Man₈/Man₄/Man₃/Man₉ ratio = 1:0.48:0.15:0.1:0.05:0.04:0.01. This demonstrates that Man₅GlcNAc₂ is the dominant glycoform present at all four glycosylation sites of OvGST1a. Besides identifying this glycoform as dominant, the analysis revealed that about 30% of the N-glycans found are smaller (Man₃ and Man₄) than those described for mammals. Interestingly, whereas the structures of Man₂GlcNAc₂ to Man₅GlcNAc₂ and the relative ratios of oligosaccharides linked to glycosylation sites 1 (N50), 2 (N79), and 3 (N134) appear to be rather similar, at glycosylation site 4 (N144) clearly larger structures, bearing Man₅GlcNAc₂ to Man₉GlcNAc₂, were detected. For further characterization of the (glyco)peptides, individual fractions from another HPLC run were collected and the amino acid sequences of all glycopeptides were confirmed by Edman sequencing. Additionally, the MALDI-TOF MS spectrum shows the protonated molecular ions of tryptic GP1_44-57 bearing two to five mannose residues (Fig. 4). Small amounts of a tryptic peptide with one missing cleavage site incorporating glycosylation site 4 (N144) were detected; this peptide bears high-mannose type glycans (Man₅ to Man₉) much larger than those found for the first glycosylation site (Fig. 4). This confirms the HPLC and ESI-MS/MS results for this site obtained for the completely digested GP4_144-156. The major components of the collected HPLC fractions were further characterized by ESI-MS/MS experiments. The daughter ion spectrum of doubly charged GP1_44-57 showed an abundance of fragment ions generated by the cleavage of monosaccharide bonds, as is explained in the fragmentation scheme of Fig. 5. In particular, the successive elimination of five hexose residues indicates the presence of a high-mannose type glycan. Peptide sequence-specific fragments from the amino and carboxy termini were much weaker but nevertheless allowed the confirmation of the peptide sequence. The other major glycopeptides were characterized in an analogous way.

View larger version (25K): [in this window] [in a new window]   FIG. 3.   Tryptic peptide mapping of the native OvGST1a and OvGST1b. The protein preparation was digested with trypsin as described in Materials and Methods. Tryptic fragments were separated by microbore HPLC, and each distinct peak was subjected to amino acid sequence analysis. For the identification of glycosylated peptides, aliquots from the fractions (asterisks) were separately analyzed for sugar constituents. Mox., oxidized methionine; RT, retention time.

View larger version (10K): [in this window] [in a new window]   FIG. 4.   Upper mass range of the MALDI-TOF spectrum of HPLC fraction 13 (compare Fig. 3) isolated by reverse-phase HPLC from OvGST1a following tryptic digestion. The peptide containing glycosylation site 1 (N50) (left site) bears truncated mannose type glycans with two to five mannose residues (M2 to M5), whereas the peptide with one missing cleavage site and containing glycosylation site 4 (N144) (right site) bears high-mannose glycans with five to eight mannose residues. From the dominant component at m/z 2841.7, a daughter ion spectrum was obtained by ESI-MS/MS as depicted in Fig. 5.

View larger version (19K): [in this window] [in a new window]   FIG. 5.   ESI daughter ion spectrum of doubly charged GP1 with five mannose residues. The major doubly charged fragment ions are due to the elimination of one to five mannose residues. Complementary glycan fragments obtained by the cleavage of the chitobiose core and the subsequent loss of the mannose residues were obtained as indicated in the fragmentation scheme. Peptide-specific fragments incorporating the carboxy-terminal (yn) or amino-terminal (bn) amino acids are marked in the spectrum. GN, N-acetylglucosamine; M, mannose.

Homology modeling of OvGST1. Alignment of OvGST1a and -1b with GSTs from various classes demonstrates the relationship to the sigma class enzymes, particularly the hematopoietic prostaglandin D synthase from rats (23), one of the products of the GST gene families in the housefly Musca domestica that is involved in pesticide resistance (57), the S-crystallins constituting the major lens proteins in squids (54), and the GSTs isolated from squid digestive glands (19).

View larger version (0K): [in this window] [in a new window]   FIG. 6.   N-glycosylation sites and the topology of the OvGST1a monomer. Shown is a ribbon presentation of a three dimensional model of OvGST1a based on the structure of the squid sigma class GST (PDB code: 1GSQ). -Helices are red, and  strands are yellow. The N-to-C direction of the structural elements can be deduced by the labeling of the secondary structures. The locations of the N glycosylation sites are orange.

Influence of N glycosylation on the recognition of OvGST1a and -1b by patient sera. To analyze the influence of the partially truncated N-glycans on the recognition of OvGST1a and -1b by the immune system of the host, 29 sera of patients with generalized onchocerciasis were tested by ELISA (Fig. 7). For the deglycosylation, the native OvGST1a and -1b were treated with N-glycanase F (Fig. 1, lanes A and C). Deglycosylation was additionally performed using endoglycosidase H (Fig. 1, lane D). This experiment was necessary to analyze the influence of the altered charge due to the replacement of amino acid N with D, caused by treatment with N-glycosidase F. The IgG antibody response to endoglycosidase H-deglycosylated OvGST1a and -1b was similar to the antibody responses measured following N-glycosidase F treatment. Furthermore, comparative ELISA with native OvGST1a and -1b with and without reheating and overnight incubation at 37°C was performed to exclude the influence of this pretreatment in the following ELISA measurements. Serological responses to native glycosylated and deglycosylated OvGST1a and -1b are indicated in Fig. 7. Levels of OvGST1a- and -1b-specific antibodies of noninfected individuals were analyzed using five European and four African control sera. In these sera, there was no recognition of the glycosylated and deglycosylated forms. The OD₄₅₀ cutoff of 0.18 can probably be attributed to conserved GST epitopes. The OD₄₅₀ values representing the IgG antibody responses to the OvGSTs are significantly higher (paired test; P < 0.05) for the native glycosylated form, i.e., the native structure that is found in the living parasite (median OD₄₅₀ = 0.66 [10th and 90th percentiles, 0.32 and 0.79, respectively]), than for the deglycosylated form (median OD₄₅₀ = 0.36 [10th and 90th percentiles, 0.19 and 0.58]) (Fig. 7). Using E. coli-expressed rOv-GST1a instead of the deglycosylated protein also led to lower IgG responses than those for the native protein (data not shown). Out of the 29 onchocerciasis patient sera tested, 2 sera did not recognize the enzymes and 2 others recognized only the glycosylated form of the enzymes.

View larger version (16K): [in this window] [in a new window]   FIG. 7.   ELISA of the IgG response of human onchocerciasis infection sera to native glycosylated and deglycosylated OvGST1s. Twenty-nine sera of patients infected with O. volvulus were tested (n = 3). The responses were significantly higher (P < 0.05) for the glycosylated form (OvGST1 plus CHO) than for the deglycosylated (OvGST1 without carbohydrates) protein. The IgG responses of the patient sera (OD450 values) are given as individual points. Boxes, 50% area of total IgG antibody responses to OvGST with and without CHO; bars,10th and 90th percentiles. The line marks the cutoff value.

Antibody recognition of the N-terminal extension of OvGST1a. When expressing the OvGST1a and -1b in E. coli, we get initiation of translation at two different methionines, resulting in the respective expression of proteins that differ in size. Whereas the 27-kDa protein has a 25-amino-acid N-terminal extension, the smaller protein does not (Fig. 2). This extension shows no homology to known GST structures. Searching the available databases did not result in any homologies to known peptide structures. By using the program ANTIGENIC (http://www.uk.embnet.org/Software/EMBOSS/) based on the method of Kolaskar and Tongaonkar (28, 44), we identified eight potential antigenic determinants in OvGST1a (A39-S52, I112-W129, G167-M184, A90-L102, L194-K206, F72-N79, S81-S88, and E147-F160) and one located in the N-terminal extension of the GST (K18-Q24). Based on this tentative information we analyzed the N-terminal extension of the GST in order to identify the antigenic potency of this OvGST1-specific region. To examine the antibody response to the N-terminal extension, 38 sera of patients with generalized onchocerciasis were tested by ELISA. For that purpose, the 27-kDa OvGST1a with N terminus (generated by mutating the second translation initiation start point from M25 to A25) was expressed and purified in order to preserve the conformational epitopes of the total protein. For comparison, we also expressed OvGST1a without the N terminus. Significantly higher antibody levels (P = 0.046) were found for OvGST1a plus the N terminus (median OD₄₅₀ = 0.28 [10th and 90th percentiles, 0.16 and 0.58, respectively]) than for the N-terminally truncated protein (median OD₄₅₀ = 0.22 [10th and 90th percentiles, 0.17 and 0.35, respectively]). Whereas four of the patient sera did not recognize the 27-kDa OvGST1a plus N-terminal extension, nine did not recognize the smaller 24.5-kDa protein. These results show that 34 out of 38 (89.5%) patient sera recognized the recombinant OvGST1a plus N-terminal extension as the antigen. From the patient sera that recognized both, i.e., the protein with and without the N-terminal extension, 22 out of 38 sera (58%) showed an enhanced reactivity to the protein with the N-terminal extension.

Reactivity of the N-terminal portion of OvGST1a with sera from patients with other helminth infections. All helminth GSTs have some distinct structural elements that lead to cross-reactivity with sera from patients infected with other helminths. For example, antibodies raised against affinity-purified OvGSTs react strongly with their Brugia pahangi and Brugia malayi counterparts (47). OvGST1a and -1b, however, each have a unique N-terminal extension not found in other GSTs so far. On account of the immunoreactivity observed, we synthesized two overlapping peptides comprising the N-terminal extension of OvGST1a and analyzed their cross-reactive properties with sera from patients infected with other helminths (L. loa, B. malayi, W. bancrofti, A. lumbricoides, T. spiralis, and S. mansoni). All of the tested sera showed low IgG responses, with OD₄₅₀ values 0.15. Twelve out of 20 sera from patients with onchocerciasis showed IgG antibody responses to the N-terminal extension of OvGST1a, with OD₄₅₀ values >0.1, and 8 of these sera reacted weakly with the two peptides (median OD₄₅₀ = 0.2 [10th and 90th percentiles, 0.052 and 0.86, respectively]; cutoff OD₄₅₀ = 0.04). None of the sera (serum pools) from patients with filarial infections with L. loa or B. malayi exceeded OD₄₅₀ values of >0.1. Two of five serum pools from patients infected with closely related filarial nematode W. bancrofti showed an IgG response to the peptides (OD₄₅₀ values of >0.1). This higher IgG reaction may be due to similar GST antigen epitopes, which might resemble the N-terminal portion of OvGST1a. As shown for the lymphatic filariasis-causing nematode B. malayi, three GSTs similar to OvGST1 and OvGST2 cross-react with anti-OvGST rabbit antisera, indicating that B. malayi possesses similar GSTs (47). The investigation of serum pools that were obtained from patients with non-filarial nematode infections demonstrates that all four pools from T. spiralis infections showed IgG responses <0.1 while one of five pools from A. lumbricoides-infected patients showed a higher IgG reaction (OD₄₅₀ = 0.12) with the N-terminal extension of OvGST1a. This may be due to a hidden or former infection with O. volvulus. The serum pools from patients infected with the trematode S. mansoni did not react with the N-terminal extension peptides (OD₄₅₀ values of <0.1).