Specific immunoglobulin measurements related to ... - NCBI - NIH

Clin Exp Immunol 1996; 106:45–54

Specific immunoglobulin measurements related to exposure and resistance to Schistosoma mansoni infection in Sudanese canal cleaners M. Z. SATTI, P. LIND*, B. J. VENNERVALD y, S. M. SULAIMAN, A. A. DAFFALLA & H. W. GHALIB z Tropical Medicine Research Institute, National Centre for Research, Khartoum, Sudan, *Danish Veterinary Laboratory and Centre for Experimental Parasitology, Royal Veterinary and Agricultural University, Copenhagen, and yDanish Bilharziasis Laboratory, Charlottenlund, Denmark, and zInstitute of Endemic Diseases, Khartoum University, Khartoum, Sudan (Accepted for publication 10 June 1996)

SUMMARY The present work comprises a longitudinal study of Schistosoma mansoni infection in occupationally hyper-exposed canal cleaners in the Sudan and the influence of chemotherapy on humoral immune parameters. The study groups included chronically infected canal cleaners (n = 19), newly recruited canal cleaners (n 17), normally exposed adults (n 31), school children (n 46) and Sudanese negative controls (n 48). Previous studies of the same canal cleaners have demonstrated that chronically infected canal cleaners were more resistant to reinfection than newly recruited canal cleaners. ELISA was used to detect specific IgE and IgG subclasses in response to whole worm antigen (WWH) and soluble egg antigen (SEA) before and 3 months after praziquantel treatment in the groups of canal cleaners and before and 1 year after treatment in normally exposed adults. When intensity of infection was correlated with IgE antibody response, the resistant group of canal cleaners (those who stopped passing ova after treatment) showed a significant positive correlation between intensity of infection and specific IgE to WWH (Spearman’s correlation coefficient 0:49, P < 0:05) compared with a highly significant negative correlation in the susceptible group (acquired new infection after treatment, Spearman’s correlation coefficient ÿ0:94, P < 0:01). Normally exposed adults and school children had significantly less specific IgE to WWH than canal cleaners, while chronically infected canal cleaners had significantly higher levels of specific IgG1 to WWH than newly recruited canal cleaners and school children, and significantly higher levels of specific IgG4 to WWH than school children. There was a significant increase in specific IgG1 and IgG4 to WWH, 3 months after treatment, in newly recruited canal cleaners and a significant decrease, 1 year after treatment, in normally exposed adults. None of the groups studied after treatment showed a significant change in their specific IgE to WWH. Normally exposed adults had significantly lower levels of specific IgE to SEA than newly recruited canal cleaners, and significantly lower levels of specific IgG1 to SEA than other infected groups. Both newly recruited canal cleaners and school children had significantly higher levels of specific IgG2 to SEA than persons in other groups. Only small differences between groups were observed with regard to specific IgG3 and IgM to SEA. Specific IgG4 to WWH and SEA showed different patterns after treatment between the resistant and susceptible groups of canal cleaners. The resistant group maintained the same level of IgG4 to WWH after treatment compared with a significant increase in the susceptible group. On the other hand, levels of specific IgG4 to SEA showed a highly significant decrease after treatment in the resistant group. In contrast, the same antibody subclass increased after treatment in the susceptible group. Generally, results show an association between IgE and IgG1 responses to WWH and resistance to reinfection. In contrast, an association was observed between IgG2 and IgM responses to SEA and susceptibility to reinfection. Keywords

canal cleaners

Mansoni hyper-exposed

resistant

susceptible

INTRODUCTION Correspondence: Dr Mohamed Ziada Satti, Tropical Medicine Research Institute, National Centre for Research, PO Box 1304, Khartoum 11111, Sudan.

# 1996 Blackwell Science

It is now generally accepted that in areas endemic for human schistosomiasis an age-dependent immunity occurs, so that after

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M. Z. Satti et al.

chemotherapeutic treatment young children are highly susceptible to reinfection, whereas older individuals, who are equally exposed to infection, are resistant [1–3]. Within endemic areas, infected individuals of all ages have high levels of circulating antibody with anti-schistosome specificity, including antibodies which react with the surface of the schistosomulum. However, the class and subclass composition of these antibodies vary with age and intensity of infection [4–6]. Since antibody isotypes differ greatly in their ability to mediate various immune effects, the isotype composition of anti-schistosome responses may have a major influence on the efficiency of antibody-dependent cytotoxic effector mechanisms, and consequently on the expression of protective immunity during schistosome infection. This finding, together with extensive demonstrations in experimental animals of the induction of protective immunity by a variety of procedures, suggests that it may eventually be possible to induce such a resistance by vaccination. This will require an appropriate protective antigen and induction of appropriate protective immune responses. Comparison of humoral immune responses and their association with protection and susceptibility in a naturally infected group, with a homogeneous exposure pattern, may be valuable in defining humoral immune parameters for protection. In order to add to the better understanding of the immunological situation of Schistosoma mansoni, the present study was carried out on selected subjects infected with S. mansoni having different degrees of exposure to the disease. A unique group which fulfils this objective is the group composed of canal cleaners. A detailed description of the canal cleaners group is given elsewhere [7]. This present study reports findings on S. mansoni infection concerning the responses to adult worm and soluble egg antigens in different immunoglobulin classes and subclasses; and ELISA tests to detect S. mansoni-specific IgE, IgM and IgG subclass antibodies have been developed. The responses in parasitologically defined groups of patients with a wide spectrum of exposure to the disease were compared. The specific objective of the study was to use data from schistosomiasis patients with different degrees of exposure to test the hypothesis that immunological responses measurable in serum samples may be related qualitatively and/or quantitatively to resistance to reinfection after chemotherapy.

SUBJECTS AND METHODS Study area and study population The study area was described elswhere [7]. The study population comprised 113 subjects. Depending on exposure to infection, the study group was divided into five groups comprising chronically infected canal cleaners, newly recruited canal cleaners, normally exposed adults, school children and uninfected controls: (i) 19 canal cleaners who manually remove the vegetation which obstructs the water flow [7], who have been working for more than 5 years; (ii) 17 canal cleaners who have been in the service for less than 1 year. The latter were newcomers to the Gezira, included to study the effect of hyper-exposure to schistosome cercariae on people without previous exposure and to provide information on acute, recently acquired infection; (iii) 31 normally exposed adults, mainly farmers, representing the normal exposure pattern; (iv) 46 school children from the age of 9 to 12 years who experience a high exposure since they normally swim in the canals; (v) 48 uninfected controls selected from outside the endemic area (Khartoum) who

were free from schistosomiasis infection. The first three groups were selected as age-matched groups (range 25–55 years).

Study design Immunological responses in Sudanese canal cleaners, with a homogeneous exposure pattern, were analysed and compared with other infected groups which were not occupationally hyperexposed. A longitudinal parasitological study was undertaken and the study subjects were followed for 1 year. Stool and urine samples were collected from the selected individuals on 3 consecutive days. Stool examination was performed on three slides/ day using the modified Kato technique [8]. A deposit of 10 ml of midday urine from each individual was examined for S. haematobium ova. The subjects were bled before and at a predetermined time after praziquantel (Biltricide; Bayer) treatment (2 2 20 mg/kg body weight) [9]. Venous blood samples of 10 ml were taken from each individual before treatment. Another blood sample was taken 3 months after treatment in the case of canal cleaners and 1 year after treatment in the case of normally exposed adults. School children were bled only before treatment. The blood was defibrinated with glass beads, centrifuged and the serum frozen in small aliquots in liquid nitrogen at ÿ1968C. The canal cleaners were treated with praziquantel at the beginning of the dry season (end of transmission season). In the absence of the transmission over time period (no re-exposure), changes in antibody levels may be assumed to be due to the effect of treatment. However, normally exposed adults were exposed to reinfection before the second follow-up bleed. Measurements of specific antibodies made it possible to assess the role of different isotypes in relation to susceptibility and resistance to reinfection in canal cleaners as evaluated by parasitological follow up. Canal cleaners with complete parasitological follow up at 3, 6, 12, and 48 weeks after treatment [7] were divided into two subgroups (regardless of their infection duration): a resistant group (n 18) which remained negative throughout, and a susceptible group (n 8) which become reinfected after praziquantel treatment. This approach identified two extreme subgroups of individual canal cleaners. Both subgroups were occupationally hyper-exposed and there was no significant difference in their pretreatment intensity. While the resistant subgroup contained 11 individuals from the chronically infected canal cleaners and seven newly recruited canal cleaners, the susceptible subgroup contained four individuals of each canal cleaners group.

Antigen preparation All stages of the parasite life cycle used to obtain antigen material were taken from a Sudanese isolate of S. mansoni which was maintained in Biomphalaria glabrata and NMRI mice at the Danish Bilharziasis Laboratory.

Soluble egg antigen Eggs were collected according to the sieve method of Dresden & Payne [10] with slight modification. Fat-free small intestines and livers of mice (7–9 weeks post-infection) were cut into small segments and left in 1.2% saline overnight at room temperature before homogenization. The homogenate was then poured into a tiered column of sieves (420 m, 180 m, 150 m, 75 m, 45 m). Eggs collected on the 45-m sieve were further washed in a Petri

# 1996 Blackwell Science Ltd, Clinical and Experimental Immunology, 106:45–54

Immunity in S. mansoni-infected Sudanese canal cleaners dish with hypertonic saline (1.7%), then put under a dissecting microscope where all debris was removed. Eggs were then rinsed in cold water and stored at ÿ708C. Tissue material retained on the 420-m sieve was further incubated overnight in the presence of trypsin and collagenase (100 mg/100 ml tissue and 10 mg/100 ml tissue, respectively), whereafter sieving and egg isolation were done as above. Eggs collected from 200 mice were homogenized in PBS in ice (pH 7.4) and then ultracentrifuged for 2 h at 100 000 g [11]. The supernatant was collected and labelled soluble egg antigen (SEA).

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(Janssen) formed a third layer, and conjugates a fourth layer (Table 1). All conjugates were incubated for 1 h at room temperature. Only interassay variations of < 10% were accepted. OPD (Sigma) substrate was used at a concentration of 0.4% (w/v) in citrate buffer pH 5 with 0.01% (v/v) H2O2. The reaction was stopped after 15 min by addition of 20% sulphuric acid. The detected optical density (OD) readings of all samples were corrected to the set reference positive according to the method of Voller et al. [14]. The discrimination level between positive and negative individuals was set at the mean of 48 normal controls + 2 s.d. above the mean [15].

Whole worm homogenate Schistosoma mansoni adult worms perfused from infected mice [12] were washed with water, homogenized in a tissue grinder on ice and centrifuged at 100 000 g for 1 h. The supernatant was collected and labelled whole worm homogenate (WWH).

Statistical analysis Spearman’s rank correlation, Wilcoxon signed rank test, one-way analysis of variance (Scheffe-range) and Mann–Whitney signed rank test were applied, where appropriate.

Protein determination for antigens The Bradford protein assay [13] was used to determine the protein concentrations of WWH and SEA.

RESULTS Parasitological results The pretreatment intensities of infection for the different study groups (geometric mean) are shown in Table 2. In a previous study on the same groups of canal cleaners, chronically infected canal cleaners were found to be more resistant to reinfection than newly recruited canal cleaners [7]. Canal cleaners and school children had statistically significant higher egg counts than normally exposed adults (P < 0:05). In addition, chronically infected cleaners showed a significantly higher egg count than school children (P < 0:05). All canal cleaners, except two individuals (6%) were passing ova before treatment, whereas 61% of normally exposed adults were passing ova. All school children were positive. When examined 1 year after treatment, 38% of those re-examined of the chronically infected canal cleaners were passing ova compared with 78% of those re-examined of the newly recruited cleaners and 26% of normally exposed adults. Schistosoma haematobium infection was not found in any of the study individuals.

Specific immunoglobulin determination ELISA was performed according to the method of Voller et al. [14] with some modifications. WWH and SEA in carbonate/bicarbonate buffer pH 9.6 were used for coating 96-well polystyrene microtitre plates (Nunc-Immuno Plate I) at room temperature overnight. One percent bovine serum albumin (BSA; Sigma Chemical Co., St Louis, MO) was then added and wells were incubated for 30 min to block the free sites of the plates. All sera were tested in duplicate and incubated overnight at room temperature. A pool of serum selected from the high responders was used as a positive control at four concentrations throughout the study. A negative serum pool was used as negative control. Optimal concentrations of antigens, sera and anti-human serum reagents determined by checkerboard titration are shown in Table 1. A four-layer ELISA system was used for IgG2 and IgG3. Monoclonal mouse anti-human IgG2 at 1:5000 and monoclonal mouse anti-human IgG3 at 1:3000

Table 1. Reagent concentrations used for specific immunoglobulin determination

Coating concentration (g/ml) WWH

IgE IgG1 "" IgG2 ~~ IgG3 ~~ IgG4 "" IgM

Dilution of serum

Dilution of conjugate

SEA

WWH

3 2

2 0.5

1 : 20 1 : 1000

1 : 20 1 : 5000

1 : 1000 1 : 10 000

1 : 1000* 1 : 5000-

ND

0.5

ND

1 : 100

ND

1 : 5000-

ND

0.5

ND

1 : 100

ND

1 : 5000-

2

1

1 : 500

1 : 1000

1 : 5000

1 : 5000-

ND

0.5

ND

1 : 5000

ND

1 : 3000*


SEA

WWH

SEA

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M. Z. Satti et al. Table 2. Egg count (eggs/g) in Schistosoma mansoni-infected groups

Egg count 3 months after treatment

Egg count before treatment

Number examined

Study group Chronically infected canal cleaners Newly recruited canal cleaners Normally exposed adults School children

Geometric mean and minimummaximum

Number examined

Egg count 1 year after treatment


Number examined


19

221 0–1832

16

5.5 0–533

16

4.4 0–342

17

148 0–1340

14

7.3 0–253

11

59 0–574

31

14 0–760 60 4–405

ND

ND

31

ND

ND

ND

3.2 0–194 ND

46

ND, Not done.

Relationship between intensity of infection and immunoglobulin in resistant and susceptible canal cleaners The resistant group showed a significantly positive correlation between intensity and IgE to WWH, whereas the susceptible group showed a significantly negative correlation between infection intensity and IgE to WWH (Table 3). The resistant group showed a significantly positive correlation between infection intensity and the IgG1 to WWH and IgG4 to WWH and SEA. The susceptible group showed a significantly negative correlation between infection intensity and IgE to SEA. A significantly negative correlation was also observed between infection intensity and IgG3 to SEA in the susceptible group. The

susceptible group showed a significantly positive correlation between infection intensity and IgM to SEA (Table 3). Antibody responses to WWH and effect of treatment IgE, IgG1 and IgG4 responses are shown in Fig. 1. Both normally exposed adults and school children had significantly less IgE to WWH than canal cleaners (P < 0:05, Fig. 1a). Chronically infected cleaners had significantly higher IgG1 to WWH than newly recruited cleaners and school children (P < 0:05, Fig. 1b). Chronically infected canal cleaners had significantly higher IgG4 to WWH than school children (P < 0:05, Fig. 1c). All groups studied had significantly higher geometric mean

Table 3. Correlation between pre-treatment intensity of infection and specific immunoglobulins in Sudanese canal cleaners

a.

Specific immunoglobulins to whole worm homogenate

Group Resistant group (n 18) Susceptible group (n 8)

IgE

IgG1

IgG4

0.49 P < 0.05 ÿ0.94 P < 0.01

0.45 P < 0.05 0.63 NS

0.48 P < 0.05 0.31 NS

b.

Specific immunoglobulins to soluble egg antigen

Group Resistant group (n 18) Susceptible group (n 8)

IgE

IgG1

IgG2

IgG3

IgG4

IgM

0.04 NS ÿ0.81 P < 0.05

0.32 NS ÿ0.52 NS

ÿ0.35 NS ÿ0.05 NS

ÿ0.22 NS ÿ0.81 P < 0.05

0.41 P < 0.05 0.40 NS

0.20 NS 0.79 P < 0.05

Values represent Spearman’s correlation coefficient and P value. NS, Not significant.


Immunity in S. mansoni-infected Sudanese canal cleaners

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Fig. 1. Specific antibody responses (before treatment) to whole worm homogenate (WWH) in schistosomiasis mansoni-infected groups: 1, chronically infected canal cleaners; 2, newly recruited canal cleaners; 3, normally exposed adults; 4, school children; 5, negative controls. The solid lines represent the geometric mean ELISA optical density (OD). The dotted lines represent the cut-off for positivity. (a) IgE. (b) IgG1. (c) IgG4.

IgE, IgG1 and IgG4 to WWH than the Sudanese negative controls (P < 0:05, Fig. 1). Both IgG1 and IgG4 determinations to WWH gave a better discrimination between infected and uninfected groups than IgE to WWH (Fig. 1). No significant change in the IgE to WWH was observed after treatment (Table 4). The IgG1 to WWH remained the same in chronically infected canal cleaners, but increased significantly in newly recruited cleaners, 3 months after treatment (P < 0:05). Normally exposed adults showed a significant drop in IgG1 to WWH, 1 year after treatment (P < 0:001) (Table 4). The IgG4 to WWH of newly recruited canal cleaners showed a significant increase 3 months after treatment, while normally exposed adults showed a significant decrease in their IgG4 to WWH response 1 year after treatment (Table 4). School children were studied only before treatment. Antibody responses to SEA and effect of treatment As shown in Fig. 2, despite their very high total IgE level (data not shown), chronically infected cleaners had only a moderate IgE to SEA response. The highest geometric mean IgE to SEA was found

in newly recruited canal cleaners and school children (Fig. 2a). However, the difference was significant only between newly recruited canal cleaners and normally exposed adults (P < 0:05). Normally exposed adults showed significantly less IgG1 to SEA than the other infected groups (P < 0:05, Fig. 2a). Both newly recruited cleaners and school children showed significantly higher IgG2 to SEA than persons in the other groups (P < 0:05, Fig. 2c). School children showed significantly higher IgG3 to SEA than normally exposed adults (P < 0:05). No significant difference was observed between other schistosomiasis groups (Fig. 2d). School children showed significantly less IgG4 to SEA than all other schistosomiasis groups (P < 0:05, Fig. 2e). There was no significant difference in IgM between the different infected groups (Fig. 2f). All groups studied had significantly higher geometric mean IgG1, IgG3, IgG4 and IgM to SEA than Sudanese negative controls (P < 0:05, Fig. 2). All groups, except normally exposed adults, had significantly higher IgE to SEA than Sudanese negative controls (P < 0:05, Fig. 2). Only newly recruited canal cleaners and school children had significantly higher IgG2 to SEA than Sudanese

Table 4. Specific immunoglobulin levels (ELISA optical density (OD)) to whole worm homogenate in Schistosoma mansoni-infected groups before and after praziquantel treatment (paired samples).

Geometric mean and rangeGeometric mean and range of IgE before of IgG1 before and after treatment and after treatment Subject group (no.) Chronically infected cleaners (n 17) Newly recruited cleaners (n 16) Normally exposed adults (n 31)

Before

0.20

After

0.22

0.07–1.10

0.08–1.19

0.24

0.26

0.05–0.81

P*

NSy

Before

0.78

After

Geometric mean and range of IgG4 before and after treatment P

0.73

Before

After

0.79

0.70

0.02–1.87

0.01–1.87

0.24

0.35

0.02–1.72

0.06–1.68

0.34

0.23

0.01–1.46

0.01–1.33

NS 0.35–1.01

0.32–1.01

0.56

0.62

0.09–0.80

0.21–0.67

0.38–0.82

0.12

0.12

0.65

0.57

0.03–0.47

0.05–0.54

0.40–0.85

0.37–0.89

NS

NS

P < 0.05

P < 0.0001

NS

*Comparison between the pre- and post-treatment immunoglobulin levels is done by Wilcoxon non-parametric test.

yNo significant difference before and after treatment.

Blood samples were collected 3 months after treatment for canal cleaners and 12 months after treatment for normally exposed adults.


P

P < 0.01

P < 0.01

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M. Z. Satti et al.

negative controls (P < 0:05, Fig. 2). IgG1, IgG4 and IgM determinations to SEA gave a better discrimination between infected and uninfected groups than IgE, IgG2 and IgG3 to SEA (Fig. 2). IgE to SEA remained unchanged after treatment (Table 5). IgG1 to SEA dropped significantly in chronically infected canal cleaners (3 months after treatment) and normally exposed adults (1 year after treatment), but was unchanged in newly recruited cleaners (Table 5). Three months after treatment, newly recruited canal cleaners maintained the same high IgG2 to SEA (Table 5). All groups studied after treatment showed a significant drop in IgG4 to SEA. The drop in IgG4 to SEA was less significant in newly recruited cleaners (Table 5). Three months after treatment, newly recruited cleaners had the highest IgM to SEA (Table 5). However, the increase in IgM to SEA after treatment was not significant. Differences in antibody responses between resistant and susceptible canal cleaners and effect of treatment Before treatment the resistant group had significantly higher IgG1 to WWH than the susceptible group (P < 0:05, Fig. 3), whereas

the susceptible group had significantly higher IgG2 and IgM to SEA (P < 0:05). IgG4 to WWH increased significantly 3 months after treatment in the susceptible group only (P < 0:05, Fig. 4). IgE and IgG1 to WWH remained without change after treatment in both resistant and susceptible groups. IgG4 to SEA dropped significantly (Fig. 4) 3 months after treatment in the resistant group (P < 0:001), whereas the same antibody increased, although not significantly, in the susceptible group. No significant change was observed 3 months after treatment in IgE, IgG2, IgG3, and IgM to SEA, either in the resistant or susceptible groups.

DISCUSSION Long-term occupational hyper-exposure of canal cleaners without adequate treatment led to a high level of infection (geometric mean (GM 221 eggs/g faeces (epg)) and irreversible pathological changes [7]. To some degree these canal cleaners have adapted to the infection. However, their work output is partially reduced and they remain a continuous source of transmission. Newly recruited

Fig. 2. Specific antibody responses (before treatment) to soluble egg antigen (SEA) in schistosomiasis mansoni-infected groups: 1, chronically infected canal cleaners; 2, newly recruited canal cleaners; 3, normally exposed adults; 4, school children; 5, negative controls. The solid lines represent the geometric mean ELISA optical density (OD). The dotted lines represent the cut-off for positivity. (a) IgE. (b) IgG1. (c) IgG2. (d) IgG3. (e) IgG4. (f) IgM.


Immunity in S. mansoni-infected Sudanese canal cleaners canal cleaners had a high infection level, though lower than chronically infected ones (GM 148 epg). This suggests that, irrespective of their previous history of treatment, chronically infected canal cleaners harboured more S. mansoni worms. The number of S. mansoni worms present is positively correlated with the level of faecal egg excretion [16]. Normally exposed adults had a significantly lower intensity of infection (GM 14 epg). Although the intensity in school children (GM 60 epg) was comparatively low, it was still significantly higher than that of normally exposed adults. These differences in intensity among the study groups may be due mainly to the patterns of exposure. Since chronically infected and newly recruited canal cleaners were similarly exposed, the difference may be due to the duration of exposure. On the other hand, since the difference in intensity between the two canal cleaner groups was not statistically significant, this may suggest the rapid establishment

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of infection in newly recruited canal cleaners despite the comparatively short duration of exposure, implying that this group was less protected. Praziquantel treatment was very effective, since the infection was cleared 3 weeks after treatment in almost all individuals, as determined by parasitological follow up [7]. One year after treatment, 38% of chronically infected canal cleaners were passing ova compared with 78% of newly recruited canal cleaners and 26% of normally exposed adults. Thus, with a similar exposure, newly recruited canal cleaners were found to be more susceptible to reinfection than their chronically infected counterparts [7]. Only two blood samples were permitted for each subject, pretreatment and 3 months after treatment for canal cleaners, and 1 year after treatment for normally exposed adults. Only pretreatment blood samples were available from school children.

Table 5. Specific immunoglobulin levels (ELISA optical density (OD)) to soluble egg antigen in Schistosoma mansoni-infected groups before and after praziquantel treatment (paired samples).

Geometric mean and rangeGeometric mean and range of IgE before of IgG1 before and after treatment and after treatment Subject group (no.) Chronically infected cleaners (n 17) Newly recruited cleaners (n 16) Normally exposed adults (n 31)

Before

After

0.29

0.26

0.10–2.80

0.07–1.53

0.63

0.56

P* NSy

NS

Before

After

0.79

0.52

0.12–1.35

0.10–1.00

0.76

0.64

Geometric mean and range of IgG2 before and after treatment P

P < 0.01

Chronically infected cleaners (n 17) Newly recruited cleaners (n 16) Normally exposed adults (n 31)

After

0.26

0.22

0.07–1.59

0.06–0.83

0.62

0.59

0.08–3.26

0.07–2.84

0.12

0.11

0.05–0.59

0.04–0.90

NS

0.26–1.54

0.32–1.88

0.08–2.28

0.08–2.13

0.38

0.26

0.16

0.14

0.13–1.28

0.09–0.76

0.03–0.74

0.03–0.63

Before

After

0.56

0.49

0.23–1.32

P

Before

After

1.27

0.99

0.21–1.20

0.03–2.37

0.02–2.14

0.65

0.63

1.10

0.90

0.20–1.55

0.19–1.77

0.29–2.04

0.44–2.31

0.50

0.42

0.82

0.52

0.18–1.21

0.15–1.05

0.01–2.43

0.01–2.24

NS

NS

P < 0.001

NS

P < 0.001

P < 0.01

Geometric mean and range of IgM before and after treatment P

P < 0.01

P < 0.05

P < 0.01

Before

After

0.64

0.49

0.23–1.65

0.21–1.93

0.52

0.61

0.24–1.05

0.24–1.28

0.41

0.31

0.19–0.99

0.11–0.82

P

NS

NS

*Comparison between the pre- and post-treatment immunoglobulin was done by Wlicoxon non-parametric test.

yNo significant difference before and after treatment.

Blood samples were collected 3 months after treatment for canal cleaners and 12 months after treatment for normally exposed adults.


P

NS

NS

Geometric mean and rangeGeometric mean and range of IgG3 before of IgG4 before and after treatment and after treatment Subject group (no.)

Before

P < 0.01

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M. Z. Satti et al.

The specific IgE response showed a strikingly different pattern between the resistant and susceptible groups. There was a significant, positive correlation between intensity of infection and specific IgE to WWH in the resistant group. In contrast, it was significant but negative in the susceptible group. The IgE response to SEA showed a significantly negative correlation with intensity of infection only in the susceptible group. These findings suggest that the same antibody responses may play different roles in different infected categories. This agrees with the findings of Hagan et al. [6] in S. haematobium and Dunne et al. [17] in S. mansoni, demonstrating a protective role for IgE. Another interesting finding in the study was the significantly negative correlation between intensity of infection and anti-SEA IgG3 in the susceptible group, suggesting a protective role of this subclass. There is no previous report of such negative correlation. However, Khalife et al. [18] demonstrated the importance of both IgG1 and IgG3 in eosinophil-dependent killing. Dafa’alla et al. [19] found a negative correlation between IgG3 and microfilarial load (live microfilaria/mg body weight) in Onchocerca volvulus, suggesting the involvement of this subclass in the clearance of the microfilariae in the tissues and leading to a significant drop in their circulation level. The significant positive correlations between intensity and IgG4 to both WWH and SEA in the resistant group suggest an important role for this subclass. The IgG4 to WWH and SEA, before and after treatment, in the resistant and susceptible groups is discussed below. Specific antibody responses showed considerable quantitative variations depending on the group studied and the antigen used. Whereas IgE to SEA were highest in newly recruited canal cleaners, both groups of canal cleaners had similar IgE levels to WWH which were significantly higher than in normally exposed adults and school children. However, the range of IgE ODs within study groups was very large, and the fact that chronically infected canal cleaners did not have the highest ODs suggests that resistance to reinfection does not relate directly to a high IgE antibody response. In a study by Jassim et al. [4] of chronic cases, a similar observation was made. The protective role of IgG1 to WWH was suggested by the fact that chronically infected canal cleaners had significantly higher levels of this antibody than newly recruited cleaners and school

children. Chronically infected canal cleaners were previously found to be more resistant to reinfection [7]. This suggests the involvement of IgG1 to WWH in protection against reinfection. No previous reports have been found about IgG1 playing such a role in schistosomiasis. However, IgG1 was found to induce a high eosinophil-mediated killing of schistosomula in vitro [18]. Recently, Auriault et al. [20] found the IgG1 response to a 28-kD antigen from S. mansoni worms to be higher in resistant than in susceptible children, although the difference between the two groups was not statistically significant. The resistant group of canal cleaners had significantly higher IgG1 to WWH than the susceptible group (Fig. 1), further indicating the involvement of this subclass in protection, and excluding the possibility that it was only a response which developed in chronic infections, because the resistant group includes individuals from both chronically infected and newly recruited canal cleaners. It seems that IgG1 to SEA plays a less important protective role, since both newly recruited canal cleaners and school children had high levels of this antibody resembling those of chronically infected canal cleaners. Newly recruited canal cleaners and school children showed significantly higher IgG2 to SEA than chronically infected cleaners and normally exposed adults. The presence of high amounts of IgG2 to SEA in newly recruited canal cleaners indicates the dominance of this subclass in recent infections, regardless of age. That this isotype blocks protective antibody responses is suggested because the first two groups were more susceptible to reinfection [3,7]. The link between IgG2 to SEA and susceptibility to reinfection is further strengthened because the susceptible group of canal cleaners, regardless of duration of infection, had significantly higher IgG2 than the resistant group. Newly recruited canal cleaners maintained the same high level of IgG2 even 3 months after treatment. School children showed significantly lower IgG4 levels to SEA than all other groups, and a significant drop in this antibody was found in all treated groups. A similar observation was previously made by Boctor & Peter [21] in Egyptian patients infected with S. mansoni: there was no change 3 months after treatment in any IgG subclasses except IgG4, where a significant drop was

Fig. 3. Differences between resistant (g, n 18) and susceptible (h, n 8) canal cleaners with respect to IgG subclass (before treatment) and IgM (after treatment) responses. WWH, Whole worm homogenate; SEA, soluble egg antigen.

Fig. 4. Effect of treatment on IgG4 response to soluble egg antigen (SEA) in resistant (n 18) and susceptible (n 8) canal cleaners. g, IgG4 to whole worm homogenate (WWH) before treatment; h, IgG4 to WWH after treatment; d, IgG4 to SEA before treatment; c, IgG4 to SEA after treatment.


Immunity in S. mansoni-infected Sudanese canal cleaners observed. The significant increase in IgG4 to WWH in newly recruited canal cleaners after treatment was surprising, as the chronically infected group, which had the highest geometric mean egg/g and accordingly the highest worm burden, showed no change after treatment. Such a rise in newly recruited canal cleaners can not simply be related to antigenic stimulation by dead worms. It is possible that IgG4 antibodies contribute to immune complexes with IgE antibody in a way that potentiates or mediates schistosomula killing without anaphylactic consequences. However, the presence of the high level of IgG4 to WWH in chronically infected canal cleaners, who were more resistant to reinfection [7], suggests that the blocking effect of this isotype may be less important than that of IgG2. Recently, high levels of IgG4 antibodies against recombinant P28 glutathione-Stransferase of S. mansoni were found to be associated with susceptibility to reinfection in children [20]. However, the susceptible group of canal cleaners maintained the same high IgG4 to SEA levels even 3 months after treatment, whereas a highly significant drop was observed in the resistant group, suggesting the association between IgG4 to SEA and susceptibility to reinfection (Fig. 2). Surprisingly, the highest geometric mean IgM level to SEA was found in chronically infected canal cleaners. However, the difference from other groups was not significant. There was a tendency to a decrease in IgM level in chronically infected canal cleaners after treatment, and a tendency to increase in newly recruited cleaners. In another study [7], the same newly recruited canal cleaners had significantly higher IgM level to cercarial antigen than chronically infected canal cleaners. However, the susceptible group of canal cleaners had significantly higher IgM to SEA after treatment than the resistant group. High IgM levels are known to be associated with susceptibility to reinfection in children [3,22]. Butterworth et al. [3,23], Butterworth & Hagan [1] and Khalife et al. [22] reported that IgM antibodies in sera of infected children will compete for the same antigen (expressed by both eggs and the schistosomulum surface) with the protective (ADCC-positive) IgG antibodies, thereby blocking the killing. Such IgM antibodies react with carbohydrate epitopes expressed both on egg polysaccharides and on the schistosomulum surface [3]. In conclusion, although many specific antibody responses were investigated, the most important observations were the link between resistance to reinfection and IgG1 to WWH and the link between susceptibility to reinfection and IgG2 and IgM to SEA. The difference in the correlation between IgE to WWH and intensity of infection in the resistant and susceptible groups suggests an important role of IgE to WWH in immunity against schistosomiasis. Studies are needed to identify the specific antigen components in such antibody responses.

ACKNOWLEDGMENTS This work was supported by the Danish International Development Assistance (DANIDA), Ministry of Foreign Affairs and Danish Bilharziasis Laboratory. Canal cleaner serum samples were collected in connection with the US AID PSTC grant no. 5.148 and NIH grant no. AI 16312. We thank Professor A. M. Elhassan for reviewing the manuscript and for his valuable comments. The support of the WHO Immunology Research and Training Centre/Lausanne-Switzerland and the National Centre for Research/ Khartoum-Sudan is very much appreciated.

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