Differential immune response to Onchocerca volvulus: IgG4 antibody responses differ in onchocerciasis patients from Guatemala and Ghana

Acta Tropica 63 (1997) 15 – 31

Differential immune response to Onchocerca 6ol6ulus: IgG4 antibody responses differ in onchocerciasis patients from Guatemala and Ghana G.E. Guzma´na,b, H.O. Akuffob,*, C. Lavebrattc, R. Luja´na a

Center for Health Studies, Institute of Research, Uni6ersidad del Valle de Guatemala, Apartado Postal 82, 01 901 Guatemala, Guatemala b Microbiology and Tumorbiology Center and Swedish Institute for Infectious Disease Control, Karolinska Institute, Box 280, S-171 77 Stockholm, Sweden c Department of Biochemistry and Biotechnology, The Royal Institute of Technology S-100 44 Stockholm, Sweden Received 3 July 1996; revised 7 September 1996; accepted 18 September 1996

Abstract Geographical differences exist in the clinical features of onchocerciasis in Central America and West Africa, which could be due in part from variations in the antigenic composition of the infecting organism. In an attempt to address this question, adult female worms of Onchocerca 6ol6ulus derived from nodules of patients from Guatemala and Ghana were compared in terms of polypeptide composition and the IgG4 antibody responses induced in patients. It was shown that a Tris-buffer soluble extract from the worms obtained in the two regions differ in polypeptide composition. Furthermore, the diagnostic polypeptides were found to be in the 30 kDa region but the recognition of these antigens was less intense and less frequently observed in the sera of microfilaria (mf) positive patients from Ghana than equivalent age and sex matched patients from Guatemala. © 1997 Elsevier Science B.V. All rights reserved Keywords: Onchocerciasis; Filariasis; Serodiagnosis; IgG4 antibodies; Onchocerca 6ol6ulus

* Corresponding author. Tel.: + 46 8 7287236; fax: +46 8 331547; e-mail: [email protected] 0001-706X/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved PII S 0 0 0 1 - 7 0 6 X ( 9 6 ) 0 0 6 1 3 - 4

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1. Introduction Onchocerciasis or river blindness is caused by the filarial nematode Onchocerca. 6ol6ulus. An estimated 18 million people in Africa and Latin America are infected by this organism. Infection can result in significant morbidity, with symptoms varying from pruritus to severe dermatological manifestations and blindness in cases of heavy and chronic infection (WHO, 1987). Theories regarding the pathogenesis of onchocerciasis, which includes inflammatory dermatitis, keratitis and chorioretinitis, suggest that clinical disease is initiated by exposure to specific parasite epitopes, but the observed spectrum of disease manifestations reflects both host and parasite factors (Mackenzie et al., 1985). Although patients with chronic filarial infection have specific antibodies distributed across all of the isotypes, the IgG4 response is remarkably elevated, accounting for up to 95% of the total IgG antibody response (Ottesen et al., 1985; Cabrera et al., 1989; Kron et al., 1993), even though IgG4 is by far the isotype with the lowest normal serum concentration. The role of the immune system in the disease process and in the control of the parasite is largely unknown and one of the major obstacles in immunodiagnosis is the cross-antigenic reactivity with closely related filarial parasites and even among other nematode species (Neppert, 1974; Lobos and Weiss, 1985). Specificity in diagnosis has been increased by detecting IgG4 instead of total IgG antibodies because of the special prominence of the former to filarial infections and because phosphocholine-containing polysaccharides from filarial parasites, and certain other cross-reactive carbohydrate antigens do not normally induce this isotype of antibodies (Ottesen et al., 1985; Lal et al., 1991; Lucius et al., 1992; Bradley et al., 1993a). There is, however, no strong evidence that IgG4 is involved in the pathology of onchocerciasis. Two forms of onchocerciasis have been reported in West Africa; savannah and rain forest type. The first indications of antigenic differences between the savannah and rain forest strains of O. 6ol6ulus were reported by Bryceson (Bryceson et al., 1976). More recently, heterogeneity between the antigens of worms from forest and the savannah was demonstrated by immunochemical techniques (Lobos and Weiss, 1985) and studies of isoenzyme patterns (Cianchi et al., 1985). The evidence indicating that different forms of the parasite exist is of particular immunological interest, in view of the suggestion that the major antigenic components in various geographic strains of O. 6ol6ulus are similar or even identical (Lucius et al., 1987). Whilst onchocerciasis is found in Africa as well as in Central America, the features of the disease differ between the two geographic regions, both in epidemiology and clinical manifestations. Furthermore, the vectors involved in transmission differ in the two regions (WHO, 1995). In Central America, onchocerciasis is distributed only in endemic foci located in the southern and eastern regions of Guatemala where the vegetation is abundant, whereas the disease is extensively spread across the whole of West Africa into parts of East Africa. The disease manifestation is generally milder in Central America than in West Africa. In Guatemala, ocular disease is rare and skin pathology, if present, is mild. In

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contrast, individuals from West Africa show differences in clinical manifestations, including severe eye complications in savannah which are absent or mild in forest areas (Anderson et al., 1974; Zea-Flores, 1985; Mackenzie et al., 1985). It is possible that the form of parasite inducing the infection may influence the nature of the host’s immune response. Furthermore, the genetic make up of the host which has been shown to influence the disease progression in other filarial diseases (Maizels et al., 1995) may also play a role in the intensity of antibody responses to O. 6ol6ulus antigens reflecting the involvement of genetic factors in the pathogenesis of onchocerciasis (Kron et al., 1993). Our study set out to investigate whether differences in the immune response to O. 6ol6ulus antigens exist between parasites from different geographic regions as a first step in investigating the observed different clinical manifestations in the two areas. Using immunoblotting techniques we analysed the immune reactivity of sera from Guatemala and Ghana to whole worm antigen in order to identify the immunodominant antigens of Guatemalan and Ghanaian O. 6ol6ulus adult female worms recognised by IgG4 antibodies. This approach measured both parameters of differences in response, i.e. antigenic make up of the infecting organism and antibody repertoire of the host.

2. Materials and methods

2.1. Study population The study groups of onchocerciasis patients were inhabitants of villages in endemic areas of Guatemala and Ghana. Onchocerciasis control had not been achieved in either region and thus there was ongoing transmission in both areas at the time of sampling. The groups under study were defined as follows, (i) microfilaria positive (mf + ): individuals from the endemic area with diagnosed skin mf with or without the presence of nodules (mf + , n=32 from each country); (ii) microfilaria negative: individuals from the endemic area with negative skin snips and no eye mf nor nodules, and no skin changes that could be attributed to onchocerciasis (mf − , n = 22 from each country); (iii) normal controls: individuals from Guatemala and Ghana with no previous history of onchocerciasis or residence in onchocerciasis endemic areas (NC, n = 10, from each country); (iv) other nematode parasite controls: Guatemalan individuals living in areas non-endemic for onchocerciasis, and infected with other intestinal nematodes, mainly Ascaris lumbricoides and Trichuris trichiura (P-GU, n =10); (v) normal European controls: Swedish individuals with no history of residence or travel to endemic areas of onchocerciasis (NEC, n = 15).

2.2. Diagnosis of onchocerciasis Parasitological examination for O. 6ol6ulus was done by four site skin snips from scapular and iliac regions and microscopic examination for the presence of mf

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following 24 h incubation of any of the skin snips confirmed the diagnosis. The arithmetic mean of the mf skin density ((MFD) in mf/mg) was calculated.

2.3. Sera and nodules Sera were prepared from clotted venous blood and kept frozen at 0 20°C until used. Non-calcified nodules were surgically excised from onchocerciasis patients under local anaesthesia and transported to the laboratory in liquid nitrogen and stored at −80°C until used. Sample collection was done according to the ethical and safety policies in the two countries, and with informed consent of the subjects.

2.4. Antigen preparation (Tris-buffer soluble fraction (TSF)) Intact non-calcified nodules were digested by the collagenase method of SchulzKey et al. (Schulz-Key et al., 1977). Female worms were washed and frozen at − 70°C in RPMI 1640 medium containing gentamycin (0.2 mg/ml). Tris-soluble antigen fractions were prepared as previously described (Lavebratt et al., 1994) and stored at − 20°C. The total protein concentration was estimated by the Bradford assay (Bradford, 1976).

2.5. One dimension sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) O. 6ol6ulus TSF antigens were separated in 15% T, 3.3% C polyacrylamide gels using the discontinuous method of Laemmli (Laemmli, 1970). After electrophoresis, the proteins were either transferred to nitrocellulose for immunodetection or stained with silver or Coomassie blue.

2.6. Two-dimensional gel electrophoresis Two-dimensional gel electrophoresis was performed as described (O’Farrel, 1975) using a Mini 2D-PAGE system (BIO-RAD). The first dimension was isoelectrofocusing (IEF) using pH 3–10 ampholites (PHARMACIA Biotech, Uppsala) and the second dimension performed on 12.5% homogeneous SDSPAGE gels. Internal two-dimensional molecular weight standards (BIO-RAD) were added to the sample for simultaneous pI and molecular weight estimation when compared to a reference gel.

2.7. Sil6er staining of proteins separated by SDS-PAGE After electrophoresis, the proteins were silver stained according to the method of Heukeshoven and Dernick (Heukeshoven and Dernick, 1985) with some modifications. Briefly, immediately after electrophoresis, the gel was immersed in fixing solution (40% ethanol, 10% acetic acid) for 30 min with gentle agitation.

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After fixing, the gel was incubated for 40 min in incubation solution (0.2% sodium thiosulfate, 4.1% sodium acetate anhydrous, 0.52% of 27% stock glutaraldehyde, 30% ethanol). The gel was washed three times for 5 min each with distilled water. After washing, the gel was left for 20 min in impregnating solution (0.1% silver nitrate, 0.02% of 37% stock formaldehyde). Silver solution was replaced with developing solution (2.5% sodium carbonate, 0.02% formaldehyde, 20% ethanol) and bands were developed with several changes of this solution. Once the bands had reached appropriate intensity, developing was stopped by leaving the gel in stop solution (1.46% EDTA (disodium salt)) for 10 min or up to 2 h. The stained gel was washed for 10 min with several changes of distilled water before photography or drying.

2.8. Electrotransfer of proteins onto nitrocellulose membranes and immunodetection The gel was equilibrated for 15 min in transfer buffer (25 mM Tris, 192 mM glycine, 10% methanol, pH 8.3) and the antigens were transferred to nitrocellulose membranes as previously described (Towbin et al., 1979). The nitrocellulose paper was blocked with 1% milk powder in 10 mM PBS, pH 7.2 for 30 min and then allowed to air-dry and stored at room temperature (20–26°C) until used. Experiments for immunodetection of Guatemalan and Ghanaian worm antigens were performed in parallel. The nitrocellulose was washed three times for 5 min in PBS containing 0.05% Tween-20 (PBST). After washing, the nitrocellulose was cut into 3 mm. The strips were incubated overnight with human serum diluted 1:500 in PBST. Strips were washed three times as described above, and incubated for 2 h at room temperature with mouse monoclonal IgG1-k anti-Human IgG4 conjugated with alkaline phosphatase (AP) (ZYMED Laboratories, San Francisco, CA; clone HP 6025) diluted 1:200 in PBST. The strips were washed five times in PBST and twice with PBS only. The bands were developed with 5Bromo-4-chloro-3-indolyl-phosphate/Nitro blue tetrazolium (FAST BCIP/NBT Tablets, Sigma, St. Louis, MO). The reaction was stopped with several washes in distilled water and the strips were allowed to air-dry in the dark at room temperature.

2.9. E6aluation and comparison of polypeptide patterns The silver stain gel was scanned using a Personal Densitometer PI (Molecular Dynamics, Sunnyvale, CA), and analysed using the computer program GelCompar 3.1R (Applied Maths, BVBA, Kortrijk, Belgium) to estimate the percentage of similarity between polypeptide profiles.

2.10. Statistical analysis x 2 analysis was used to evaluate the statistical significance of the relationship between the presence of mf in skin and the recognition of O. 6ol6ulus antigens.

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3. Results The villages from where the sera were taken were comparable in terms of the socio-economic situation. None of those from whom serum was taken were under-nourished as judged by the physical examination they underwent. There was ongoing transmission of O. 6ol6ulus in both areas at the time of sampling and worms from only non-calcified nodules were used for antigen preparation. Sera from 32 mf + and 22 mf − individuals from Guatemala were selected on the basis of age and sex in order to match an equal number of Ghanaian sera. All except one patient’s sera were matched on such basis. Table 1 shows the age and sex distributions and the MFD in four skin snips expressed as the arithmetic mean (Lucius et al., 1987; Kron et al., 1993). The majority of the Guatemalan individuals were co-infected with gastrointestinal parasites A. lumbricoides and T. trichiura. Fifteen of the Ghanaian individuals were also co-infected with other parasites (S. stercoralis, A. lumbricoides, and hookworm). The age and sex distributions of Guatemalan and Ghanaian groups were comparable.

3.1. Electrophoretic polypeptide patterns of Guatemalan and Ghanaian TSF Fig. 1 shows the electrophoretic patterns of Guatemalan and Ghanaian TSF with equal amounts of protein loaded on a gradient gel. Since the apparent absence of some high molecular weight bands in TSF-GU could have been the result of very small amounts of those proteins in the sample, electrophoresis was carried out loading double the amount of TSF-GU. Doubling the amount of TSF-GU resulted in an increase in the intensity of staining of the high molecular weight bands, whereas no alteration, neither in colour nor intensity, was observed in the other parts of the polypeptide profile (Fig. 1). Ten individual worms from different nodules from Guatemala and 12 from Ghana were prepared and analysed. Worms from Guatemala showed similar electrophoretic patterns within their group, and amongst the Ghanaian worms the patterns were also similar (data not shown). Thus in subsequent experiments pools of TSF from each group were used. The comparison of the polypeptide profiles of Guatemalan and Ghanaian TSF revealed differences in the staining pattern between the two preparations. The most marked Table 1 Age and sex distribution of Guatemalan (GU) and Ghanaian (GH) microfilaria positive and microfilaria negative individuals AGE (years)

Guatemalan mf+ Guatemalan mf− Ghanaian mf+ Ghanaian mf−

SEX

MFD (mf/mg)

Range

Mean 9S.D.

Males

Females

Range

18–73 18–59 18–74 18–60

43.7 911.2 31.1 9 10.6 43.6 911.2 32.9 9 10.5

15 12 16 12

17 10 16 10

0.26 – 236.6 0 0.1 – 151.25 0

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Fig. 1. Silver stained polyacrylamide gradient (10 – 17.5%) gel showing the electrophoretic polypeptide patterns of TSF-GU and TSF-GH. LMW, low molecular weight standards. Arrows indicate the main visual differences between the two polypeptide profiles. The percentage of similarity between the indicated sectors along the polypeptide profiles is given on the right, as calculated by GelCompar3.1R.

differences were observed along the 30–94 kDa region (Fig. 1). Along this region, several doublets of bands were observed in TSF-GH but not in TSF-GU. One of the most prominent bands in TSF-GH (45 kDa apparent molecular weight) and the doublet of sharp bands immediately below were not present in TSF-GU. Instead, there were two more diffuse bands of 39 and 46 kDa. One band of 37 kDa in TSF-GU was apparent instead of bands of 38 and 37 kDa in TSF-GH. Likewise,

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a sharp band of 33 kDa in TSF-GU appeared instead of a doublet located at 34 kDa in TSF-GH. The eletrophoretic pattern of polypeptides around 30 kDa in the two preparations were markedly different. In this region, the bands in TSF-GU were broader and less in number than those in TSF-GH. There were also common bands in both TSF preparations, the most prominent with an apparent molecular weight of 23 kDa. Analysis of the gel using GelCompar3.1R showed that the two polypeptide profiles were less than 30% similar. The polypeptide profiles were 75% similar between 45 kDa and above, whereas between 25 and 45 kDa the similarity was only 29% (Fig. 1). The TSF-GU and TSF-GH showed different staining characteristics following silver staining. Using this staining technique a yellow or yellow-orange colour is characteristic of highly acidic, highly basic or glycosylated proteins (Nielsen and Brown, 1984). The main differences in staining were observed in the 30–43 kDa region, where TSF-GU showed yellow or yellow orange bands whereas TSF-GH showed not only darker but sharper bands. The common polypeptide of 23 kDa was yellow during the first seconds of the staining, and turned yellow-orange by the end of the developing step. Two-dimensional gel electrophoresis of Guatemalan and Ghanaian TSF confirmed the findings of the one-dimensional electrophoresis and showed that, although the major polypeptide pattern was similar for the two extracts, differences were also apparent. The greatest differences were observed in the polypeptides resolved between 30 and 67 kDa (data not shown).

3.2. Reproducibility of the immunoblots Four sera from Guatemala and four from Ghana, were tested on two separate occasions to assess the reproducibility of the immunoblot. Reproducible patterns of immunostained bands were obtained in all instances (data not shown).

3.3. IgG4 antibody recognition of antigens in Guatemalan TSF (TSF-GU) The general IgG4 antibody pattern of antigen recognition of TSF-GU by the age and sex matched Guatemalan and Ghanaian onchocerciasis sera was essentially similar although some obvious differences were also noted (Fig. 2). Sixty-one percent of Guatemalan mf + sera showed a more intense response against TSF-GU than Ghanaian mf + sera, Fig. 3A. While the opposite was true in 29% of sera compared (Fig. 3C). Comparable intensities were seen in 10% of the matched sera (Fig. 3B). Microfilaria positive Guatemalan and Ghanaian sera consistently recognised two groups of antigens of 20 and 30 kDa (Fig. 3). The group of antigens at 30 kDa was recognised as a broad band made up of, at least, three separate bands, the most prominent of which was of approximately 33 kDa, while the group of antigens at 20 kDa was recognised as a more diffuse broad band, consisting of, at least, two bands. Some individual Guatemalan and Ghanaian sera detected other minor antigens in the high and low molecular weight regions. Control sera from individuals outside the endemic area showed no response against TSF-GU antigens.

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As shown in Table 2, the majority of mf + sera from both groups (97 and 74%, Guatemalan and Ghanaian, respectively) reacted against the major group of antigens at 30 kDa. Significantly more Guatemalan mf + sera recognised the 30 kDa antigens in TSF-GU than the corresponding matched Ghanaian sera (PB-

Fig. 2. Representative blots of various groups of Guatemalan (odd numbers) and Ghanaian (even numbers) onchocerciasis sera reacting against TSF-GU (top) and TSF-GH (bottom). The position of the molecular weight standards is indicated on the left. NC-GU, normal controls from Guatemala; NC-GH, normal controls from Ghana; P-GU, other parasite controls (mainly A. lumbricoides and T. trichiura); NEC, normal European controls.

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Fig. 3. Representative blots of age and sex matched Guatemalan (GU) and Ghanaian (GH) mf + sera showing different intensities in the recognition of TSF-GU and TSF-GH antigens. A, higher intensity of response in Guatemalan mf + sera than in the corresponding Ghanaian matched sera; this kind of pattern was observed in 19/31 (61%), of the paired sera (age, range 18 – 73). B, comparable intensity of response of Guatemalan and Ghanaian mf + sera; 13% of the pairs tested showed this pattern (age, range 40–45). C, lower intensity of response in the Guatemalan mf + sera compared to the corresponding Ghanaian matched mf + sera; 29% of the mf + pairs tested showed this kind of pattern (age, range 30–64). The position of the 30 and 20 kDa antigens, the MFD in mf/mg, and the presence of nodules is indicated.

0.05). These antigens were also detected by 41% of Guatemalan and 45% of Ghanaian mf − sera. Significantly more Guatemalan mf + sera (84%) reacted against the 20 kDa antigens than the corresponding matched Ghanaian sera (48%) (P B 0.01). A few mf − sera from both groups showed positive reaction against these antigens; 27 and 18% of Guatemalan and Ghanaian sera, respectively. The majority of Guatemalan mf + sera (84%) recognised both the 30 and 20 kDa antigens whereas only 52% of Ghanaian mf + sera showed a positive reaction against both antigens (P B0.01). Twenty eight percent of Guatemalan and 18% of Ghanaian mf − sera reacted against both, 20 and 30 kDa antigens, but this difference was not statistically significant. In general, the same sera that recognised the 30 kDa antigens also showed positive reaction against the group at 20 kDa as well; the opposite situation was not necessarily evident.

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There was a strong relationship between recognition of 20 and 30 kDa antigens, separately or in combination, in TSF-GU and the presence of mf in skin in the Guatemalan and Ghanaian mf + sera (PB 0.001).

3.4. IgG4 antibody recognition of antigens in Ghanaian TSF (TSF-GH) Despite the observed variation in the polypeptide distribution in the TSF-GU and TSF-GH, the main immunostained bands of TSF-GH were similar to those of TSF-GU. The intensity of response could also be divided into three groups as for the TSF-GU (Fig. 3A – C). These three patterns of intensity in the IgG4 response were observed in essentially the same Guatemalan and Ghanaian sera that reacted against TSF-GU. In contrast to the recognition pattern observed in TSF-GU, the multiple antigens at 30 kDa in TSF-GH were recognised as three separate bands by mf + sera; some reacted against all the three whereas others only reacted against one or two. The recognition of antigens at 20 kDa, was as a group of, at least, three separate bands. None of the control sera showed reactivity against any of these antigens present in TSF-GH. Significantly more Guatemalan mf + sera (81%) recognised the 30 kDa antigens than the corresponding matched Ghanaian sera (52%, PB 0.05) (Table 2). Fifty percent of the Guatemalan and 68% of the Ghanaian matched mf − sera detected these polypeptides. The antigens at 20 kDa were recognised by 45% of Guatemalan and 39% Ghanaian mf + sera, as well as 32 and 18% of Guatemalan and Ghanaian mf − sera, respectively. Forty-five percent of Guatemalan and 39% Ghanaian mf + sera, 32 and 18% of Guatemalan and Ghanaian mf − sera, respectively, reacted against both the 20 and 30 kDa antigens. Table 2 Most prominent Onchocerca 6ol6ulus antigens recognised by IgG4 antibodies from individuals in endemic onchocerciasis areas from Guatemala and Ghana Guatemalan sera MW (kDa) mf+ Guatemalan 30 20 20 and 30

TSF 30/31 (97) 26/31 (84) 25/31 (81)

Ghanaian TSF 30 25/31 (81) 20 14/31 (45) 20 and 14/31 (45) 30

Ghanaian sera

Control sera

mf+

mf−

NC-GU

NC-GH

P-GU

NEC

9/22 (41) 6/22 (27) 6/22 (27)

22/31 (74) 15/31 (48) 15/31 (48)

10/22 (45) 4/22 (18) 4/22 (18)

0 0 0

0 0 0

0 0 0

0 0 0

11/22 (50) 7/22 (32) 7/22 (32)

16/31 (52) 12/31 (39) 12/31 (39)

15/22 (68) 4/22 (18) 4/22 (18)

0 0 0

0 0 0

0 0 0

0 0 0

mf−

Numbers in parenthesis represent the percentage of sera in each group responding to the antigens. NC-GU, normal controls from Guatemala; NC-GH, normal controls form Ghana; P-GU, other parasite controls (only from Guatemala); NEC, normal European controls (from Sweden).

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Guatemalan mf + sera also showed a strong relationship between recognition of the 30 but not the 20 kDa antigens in TSF-GH and the presence of mf in skin (P B 0.05). Such a relationship was not observed within the Ghanaian sera under study, neither for the 20 nor the 30 kDa antigens.

3.5. Sensiti6ity and specificity estimations Since the 30 kDa antigens appeared to be relevant regarding the IgG4 response, sensitivity and specificity were estimated using the response to these antigens. Sensitivity using TSF-GU and Guatemalan mf + sera was 97 and 74% when using Ghanaian mf + sera for the estimation. Sensitivity using TSF-GH and Guatemalan sera was 81%, whereas 50% was obtained using Ghanaian sera for the analysis. Specificity for O. 6ol6ulus was 100% using TSF-GU or TSF-GH, since none of the control sera (P-GU, NC, and NEC) gave a positive reaction against the 30 kDa antigens.

4. Discussion We have compared the polypeptide composition of worms derived from Guatemala and Ghana and the antibody response to these worms in a group of well characterised age and sex matched sera. We show here that using both, one- and two-dimensional electrophoresis, Tris-buffer soluble extracts of the female adult worms from the two regions differed in polypeptide composition. However, the antigenic recognition of the polypeptides was essentially similar between the extracts from Guatemala and Ghana (TSF-GU and TSF-GH), although the detailed recognition patterns around the 30 kDa region differed. Our results also showed that the diagnostic polypeptide recognition was mainly in the 30 and not in the 20 kD region and the IgG4 response to this group of antigens was more frequently observed in the sera of the age and sex matched Guatemalan patients than the sera from the Ghanaian patients. An apparent absence of bands in the high molecular weight region was observed in the TSF-GU polypeptide profile. These proteins were difficult to stain (yellow-orange in colour) and therefore, gave the impression of absence of material when the gel was photographed. This phenomenon was confirmed in the second dimension electrophoresis where a great number of spots stained very weakly whereas others showed an optimal intensity of staining. The relative abundance of yellow-orange proteins in TSF-GU, observed in both one- and two-dimensional gels, could indicate the presence of very basic proteins, difficult to stain with the silver nitrate method used here. The chromatism exhibited by basic proteins has been described and explained in terms of differences in protein-silver ion complex formation, related to the amino acid composition (Rabilloud et al., 1994). Thus TSF-GU may contain more basic components than TSF-GH. Since the TSFs were prepared in the same way, utilising only EDTA to inhibit proteases in both cases, any differences observed between TSF-GU and TSF-GH are likely due to the variability in the

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starting material. Only minor biochemical differences between isolates of O. 6ol6ulus from the savannah and rain forest were obtained with one-dimensional SDS-PAGE (Lobos and Weiss, 1985; Lucius et al., 1987), although the pathological picture induced by these two strains are distinct. Using the same technique we show that a pool of ten extracts with very similar protein patterns derived from worms from Guatemala differed markedly at this level from a similar pool of worm extracts from Ghana, supporting the idea of geographic diversity of the parasite. Clearly, more extensive epidemiological studies are required to confirm this hypothesis. Studies addressing the evolutionary history of O. 6ol6ulus using DNA sequencing have demonstrated significant differences between the Forest and Savannah strains of Africa (Zimmerman et al., 1994). Interestingly, parasite populations from the New World (Guatemala and Brazil) were indistinguishable from the Savannah strains. The parasite population from Ghana used in the present study was derived from the forest area and thus distinct differences between these and Guatemalan strains may be expected. The close evolutionary identity between the Guatemalan worms and the severe disease causing Savannah strain would suggest a governing role of the host immune response in the final course of the infection. Despite the marked differences in their polypeptide composition between the TSF-GU and TSF-GH the immunodominant components of both preparations migrated at the same position within the electrophoretic patterns (Fig. 3), although the intensity of the bands and the recognition pattern observed with the Western blot technique differed. Whilst the immunodominant antigens (at 30 and 20 kDa) in TSF-GU were recognised as broad bands, the immunodominant components in TSF-GH (at 30 kDa) were detected as a set of up to three separate and sharp bands. An immunodominant antigen of 33 kDa with a degradation product of 20 kDa (Ov33) has been described and shown to be diagnostic for onchocerciasis (Lucius et al., 1988a,b, 1992; Tawill et al., 1995). In the present study, two antigens at 30 and 20 kDa were diagnostic for onchocerciasis when sera from Guatemalan patients were used, whilst only the 30 kDa was diagnostic using the Ghanaian patient sera. Broad immunostaining patterns often indicate the presence of glycoproteins and this observation, together with the electrophoretic pattern observed (Fig. 1) suggests that the immunodominant antigens at 30 kDa in TSF-GU are glycosylated polypeptides. In contrast, the immunostaining pattern of the immunodominant antigens in TSF-GH and their electrophoretic pattern as detected by silver staining, suggest that these polypeptides are less glycosylated (if at all) than the Guatemalan antigens. Such differences between the protein glycosylation in this molecular weight region, as well as the variation in immunostaining patterns could be due to changes at the transcriptional, translational or post-translational level, as previously discussed (Lobos and Weiss, 1985). This issue is being currently addressed through other biochemical approaches. Three patterns of intensity in the IgG4 response were observed to both, TSF-GU and TSF-GH by the respective sera. This suggests that these varying patterns were not due to differences in TSF preparation. The most frequently observed pattern was characterised by higher intensity of response within the Guatemalan mf + sera,

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than the matched Ghanaian mf + sera. Differences in antibody responses against O. 6ol6ulus by different ethnic groups have been reported in Ecuadorian Indians and Blacks (Kron et al., 1993). In that study, Amerindians demonstrated a more intense IgG response and more frequent recognition of low molecular weight parasite antigens than the Blacks of African origin from the same area. In addition, significantly higher levels of immunoglobulins against O. 6ol6ulus were demonstrated in the Amerindians, compared to the Black population. The frequency of recognition of the 30 kDa antigens was higher in Guatemalan mf + sera using homologous or heterologous TSF. A higher proportion of Ghanaian mf + sera recognised the 30 kDa antigens in the Guatemalan rather than the homologous TSF. There was a positive relationship between the recognition of these antigens and the presence of mf in skin. IgG4 antibodies to the proteins of the adult worm were produced in the Ghanaian patients, but these antibodies reacted more efficiently with the proteins of the Guatemalan TSF, which appeared to differ in chemical composition from TSF-GH. The mf load, however, did not reflect the intensity of the IgG4 response in contrast to findings in studies made in Sudan (Daffa’alla et al., 1992). Recently, a glycoprotein (Ov20) was isolated, sequenced and the native antigen encoded by the cDNA characterised. Recognition of this glycoprotein was shown to be diagnostic for onchocerciasis (Tree et al., 1995) and thus has been included in the WHO tri-cocktail being evaluated as a serodiagnostic tool (Bradley et al., 1993b). Our 20 kDa antigen was only appreciably recognised by the Guatemalan mf + sera on the homologous TSF. If variations in antigen-antibody interactions are influenced by the extent of glycosylation or acidic or basic nature of the antigenic proteins, this could have important consequences on the choice of defined proteins for use in diagnosis of onchocerciasis. Sensitivity using the 30 kDa antigens as markers was above 80% when Guatemalan but not the Ghanaian sera were used for the analysis on TSF-GU or TSF-GH and the specificity was 100% for both TSF-GU and TSF-GH using sera from normal controls or sera from individuals with other worm infestations. The use of IgG4 responses, which have been shown to enhance the specificity of onchocerciasis diagnosis (Ottesen et al., 1985; Lucius et al., 1992), may not be as suitable for use in one geographical area as in another since only 52% of mf + Ghanaian sera had measurable IgG4 response to the homologous TSF and those responses tended to be weaker than the Guatemalan sera response. We and others, using detergent-soluble extracts or semi-purified fractions of the adult worm have previously reported low sensitivity values when the IgG4 response was evaluated in populations from West Africa (Egwang et al., 1994; Lavebratt et al., 1994). Furthermore, low IgG4 levels have been shown to occur in as many as 10% of microfilaremic persons (Marley et al., 1995). The complex antigens of the O. 6ol6ulus worm induce both humoral and cellular responses in the host, the response to many of which may be genetically restricted. The final clinical outcome following O. 6ol6ulus infection involves all these parameters and is as yet not well understood. There is little evidence relating IgG4 levels and pathology (Daffa’alla et al., 1992), but differential antibody response (class and subclass) to variable O. 6ol6ulus worm antigens could influence the disease expres-

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sion. For example, differential IgG3 responses to onchocercal antigens is seen in the various clinical manifestations of onchocerciasis. IgG3 responses to a group of antigens have been associated with endemic controls (Cabrera et al., 1988), but the same subclass expressed at high levels is evident in the hypereactive sowda form of onchocerciasis, found primarily in the Arabian peninsula and Africa. The mechanisms involved in the apparent lowered cellular responsiveness to onchocercal antigens in generalised onchocerciasis (Greene et al., 1983; Elkalifa et al., 1991; Ottesen, 1995) are unclear although the possibilities of distinct down regulatory antigens have been suggested (Akuffo et al., 1996). A further variable which could influence the antigenic nature of O. 6ol6ulus and in turn the host immune response, is the vector involved in transmission of the parasite. However, the role of this factor has not been explored. In this study we do not correlate distinct antigenic patterns of O. 6ol6ulus adult worms with differential clinical manifestations of onchocerciasis. We do, however, show that differences in the polypeptide composition do exist between worms derived from nodules from parasites in Guatemala (Central America) and Ghana (West Africa). We also show that the IgG4 response to O. 6ol6ulus antigens was less frequent in the sera from Ghana. The implications from these studies suggest that the nature of the antigens used for diagnosis and the antibody class detected may need to be evaluated in different geographic regions before extensive use.

Acknowledgements This work is part of the M. Sc. thesis of G. Guzma´n and has been supported by SAREC (Swedish Agency for research Co-operation with Developing Countries) through KIRT (Karolinska Institute Research and Training Program), and CHS (Center for Health Studies, Institute of Research, Universidad del Valle de Guatemala, Guatemala, CA). The work reported here was also supported in part by the United States Agency for international Development (USAID) Specific Support Grant No. DPE-5542-G-SS-7033-00, Innovative Scientific Research Project No. 936-5542.

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