Eur. J. Immunol. 1991. 21: 1801-1807

Chum-Bo Xu, Claudie Verwaerde, Jean-Marie Grzych, Josette Fontaine and Andr6 Capron

Centre d’ImmMologie et Biohgie Parasitaire, Unit6 Mixte INSERM U 167 - CNRS 624, Institut Pasteur, Lille

Immune effects of anti-Sm 28 GST mAb in S. mansoni

1801

A monoclonal antibody blocking the Schistosoma nulasoni 28-kDa glutathione S-transferase activity reduces female worm fecundity and egg viability” The protective effects of two different monoclonal antibodies (mAb) raised against the Schistosoma mansoni 28-kDa glutathione S-transferase (Sm 28 GST) were investigated. l h o mAb of the same isotype (IgM) have been selected according to the blocking effect on Sm 28 GSTenzymaticactivity (S13) or the lack of blockade (H12).When passively transfered into Fischer rats, both S13 and H12 significantly reduced the worm burden. In BALBlc mice clear effects on female worm fecundity and egg viability were observed when the S13 mAb was transfered; these effects included significantly reduced loads of intestinal eggs, reduced egg hatching rates and an increased proportion of non-living eggs. No effect on egg production and egg hatching was observed in H12-treated mice. In addition, worm pairs recovered from S13-but not H12-treated mice laid significantlyfewer eggs in vitro, and normal worm pairs incubated in virro with the S13mAb produced significantlyfewer eggs than those incubated with H12 mAb. The impairment of egg hatching ability was also reproduced in vitro by the S13 mAb. These data suggest the existence of two different effector mechanisms induced by immunization with Sm 28 GST. The effect on the schistosome worm burden appears to be independent of GST activity whereas the effect on S. mamoni female fecundity and egg viability seems to be significantly linked to the inactivation of the enzymatic site.

1 Introduction Schistosomiasis is a world-wide parasitic disease which affects about 200 million people and several species of domesticated animals, such as cattle, buffalo, sheep and goat, in the tropical areas of developingcountries. Despite the progress of various snail control programs and the development of a highly efficient drug, praziquantel, the spread of this disease has not been significantly reduced. It is agreed that chemotherapy alone does not prevent reinfection. Moreover, the reported resistance of Schistosoma mansoni to oxarnniquine [I] suggests that the wide, sometimes even excessive usage of anti-schistmomd chemicals may favor the emergence of drug-resistant strains of schistosomes. In this context, an effective vaccine strategy is an indispensable addition to current methods, in order to achieve control of the infection.

protonephridiai cells and subtegumental parenchymal cells of S. mansoni [2,3]. It has been shown to induce protective immunity in a variety of animal models, such as mice, rats, hamsters [4,5]and baboons [ti]. Since its identification, this protein has been cloned, sequenced and expressedin E. coli [ 5 ] . In addition, the major epitopes responsible for eliciting Tand B cell responses have been identified in rats and mice [71.

Another interesting aspect of this potential vaccine candidate lies in its glutathione S-transferase (GST) activity [3]. GST form a large multigenic family of isoenzymes which are generally considered to play a major role in detoxifying various kinds of electrophilesand oxidative-freeradicals by a reduction process dependent on reduced glutathione (GSH; [S]). It was thus tempting to speculate that schistosome GST may play a key part in the survival mechanisms of schistosomes against host protective immunity. In this The 28-kDa protein (refered to as Sm 28 GST) is an antigen study, a mAb directed against the 28-kDa antigen which present in the schistosomular and adult worm tegument, blocks the expression of its GST enzymatic activity was identified.We have i stigated whether this property may participate in the ctive effects of anti-Sm 28 GST antibodies toward schistosomeinfection, in terms of worm [I 94131 survival, egg laying and egg viability and, therefore, in the * This work was accomplished With the financial support from the elimination of the parasite. Commission of Science, Technology and Development, European Community (Brussels) for C-€3. Xu as a post-doctoral fellow.This work was also supported by INSERM (U167), C N R S (UA 624) and the Edna McConnell Clark Foundation.

Comspodenee: Chuan-Bo Xu, Centre d’Immunologie et de Biologie Parasitaire, Institut Pasteur, 1 rue du Professeur A. Calmette, 59019 Lille a d e x , France

2 Materials and methods 2.1 Animals and parasites

Seven- to eight-week-old male Fischer rats and fourweek-old BALB/c mice were used in the passive transfer AbbreoPatioss: CDNB: l-Chloro-2 ,Cdinitrobenzene GSH: experiments. The Schistosoma mansoni life cycle was Glutathione GSR Glutathione S-transferase Sm 28t Schisfoso- accomplished employing hamstem as definitive, and Biomphalaria glabrata as intermediate hosts. ma mamoni 28-kDa Ag 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991

+

OOl4-2980/9l/08oS-lS1$~ S O .25/0

1802

C.-B. Xu, C.Verwaerde,J.-M. Grzych et al.

2.2 Antigens and antisera

Recombinant Sm 28 GST was produced in vitro by expression of the corresponding cDNA clone in E. coli by Transgltne S. A. (Strasbourg,France). For the purification, the soluble extract (10 min, 20000 x g) from E. coli was applied to a 7 ml S-linked GSH-agarose affinity column (Sigma, Poole, GB). The GST fraction was eluted with , 9.1) containing 7 mM GSH and Tris-NaOH (50 m ~ pH 0.1 m~ D l T [9].The eluate was dialyzed overnight against 10 m~ potassium phosphate buffer, pH 7.0 and concentrated on an Amicon membrane (PM 10, Danvers, MA). The protein content of the GST fraction was determined by the Lowry method using BSA as a standard [lo].

Eur. J. Immunol. 1991. 21: 1801-1807

overnight. After saturation in PBS containing 2% (w/v) BSA (Fluka Chemie AG) and three washes in PBS containing 0.1% Tween-20 (Prolabo, France), which was used throughout this test for all the washes between steps, immune sera or mAb ascites fluids were added at a dilution of to and incubation was performed at 37 "C for 2 h. Another 2-h incubation with peroxidase-conjugated rabbit anti-rat IgG (H+L) (ICN ImmunoBiologicals, GB, final dilution 1:2000) was carried out at 37°C before addition of the substrate o-phenylenediamine (OPD, Sigma, St. Louis, MO) and absorbance values were read at 492 nm (Behring ELISA Reader).

2.5 GST inhibition test Native total S. mansoni GST was prepared from adult schistosomes. Worms were homogenized in equilibration The GST catalyzed reaction was carried out using buffer (10 m~ potassium phosphate, pH 7.0) containing l,-chloro-2,4-dinitrobenzene(CDNB; Sigma) as a sub1.15% (w/v) KCl, 0.5 mM PMSF and 1mM EDTA by strate according to Habig et al. [14]. Sm 28 GST (0.5 pg) employing an ultra-turrax homogenizer. The homogenate was incubated with serum-freemAb cell culture SN (50,100 was ultrasonicated twice in ice and then spun at 10OOO rpm or 200 pl), rat mAb ascites fluids (2,5, 10,20 or 40 pl), or for 20 min at 4°C (Beckman Model 2 MK centrifuge).The with purified IgM (1, 5 and 8 pg), for 1h at room SN represented total schistosome antigens.The extraction temperature and for another 4 h at 4°C. After incubation, of GST from this antigen solution was performed as stated the enzymatic reaction was performed in 0.8 ml potassium , 6.5) with 5 m~ GSH and phosphate buffer (50 m ~ pH above for recombinant GST. 1m~ CDNB.The absorbance was measured at 340 nm in a The anti-schistosome total GST and anti-Sm 28 GST sera spectrophotometer (DUB-64, Beckman) every 30 s for up were prepared as previously described [4]. Two-month-old to 6 min. Similarenzyme inhibitiontest was carried out with male Fischer rats were immunized S.C. with 100 pg total or rat liver GST purchased from Sigma (75% of purity, Sm 28 GST Ag in an equal volume of CFA (Difco, Detroit, Sigma). MI).They were boosted twice, 3 and 5 weeks later with Ag in IFA (Difco).The control rats were injected with adjuvant only. Normal rat serum (NRS) was obtained from non- 2.6 Passive transfer experiments immunized rats. 2.6.1 Rat model 2.3 mAb

Fischer rats were exposed to lo00 cercariae [15]. Passive The mAb used were produced by hybridization of splenic transfer i.v. of 2 ml ascites fluids containing 2 mg mAb was lymphocytes from rats immunized with adult worm 28-kDa done 4 h later. The parasite burden was evaluated 20 days Ags with rat myeloma IR 983 F (named IR lin this report) postinfection by liver perfusion [15]. As an indicator of according to the protocol previously reported [ll]. Two protection, the worm burden reduction (R) was calculated mAb of IgM isotype, S13.38 and H12.7 (named S13 and by the following formula: H12, respectively), that recognized Sm 28 GST were selected according to their capacity to inhibit Sm 28 GST R Yo = (A-B)/A X 100 enzyme activity. S13 was able to inhibit Sm 28 GSTactivity while H12 was not. IA2-23 and C3-109 (named IA and C3, where, A is the average worm recovery of the control group respectively), which were produced by the hybridomas of and B is the group of rats treated with S13 or H12 splenic lymphocytes from LOU/C rats immunized with mAb . Onchocercu volvulus soluble antigens [12] and with S. mansoni glycoprotein-enriched fraction, respectively, along with IR, were employed as irrelevant IgM mAb controls. 2.6.2 Mouse model After the production, ascites fluids were further purified to obtain an IgM fraction first by two steps of precipitation BALB/c mice were infected with 100 cercariae [15]. mAb with 40% (w/v) ammonium sulfate [13] and then byACA34 (0.15 ml containing 0.15 mg Ab per mouse) was injected i.v. Sepharose (UltrogeP, LKB, Bromma, Sweden) gel filtra- or i.p. by either one injection at either day 0 or day 30, or tion. The IgM peak eluting at the exclusion volume was two injections at day 0 and day 15.Whole body perfusion concentrated with Acquacide 111(flake polyethyleneglycol, was carried out 45 to 50 days post-infection. The small Calbiochem, La Jolla, CA) and the protein content was intestines were also collected and kept in ice for estimations of egg numbers. measured [101. 2.4 ELISA

2.7 Analysis of egg production

The plates (96-well, Nunc, Roskilde, Denmark) were coated with Sm 28 GST protein (0.1 pg/well) at 4°C

Schistosomeadult pairs were cultivated in vitro as previously described [16]. Adult worms were sterily perfused from

Eur. J. Immunol. 1991.21: 1801-1807

Immune effects of anti-Sm 28 GST mAb in S. rnansani

the portal vein of hamsters infected with S. mansoni 40 days before. The paired worms were washed in RPMI 1640 supplemented with 20% FCS, 0.1% glucose, and 5 x M 2-ME. Three pairs of worms were collected and cultured in a 25 cm2 tissue culture flask (Corning Glass Works, Bagneaux-Sur-Loing, France) in 5 ml of the same medium complemented with SRBC (Biolyon, Dardilly, France) at a final concentration of 108celldml at 37 "C and 5% C02.The inactivated (56"C, 30 min) mAb (S13, H12 or IA) ascites fluids (100 and 200 pl) or purified IgM (10, 20 and 40 pg) were added to the cultures. A group was kept without adding any mAb as a negative control. Half of the medium of the cultures was carefully changed every 2 days after the establishment of the culture and half doses of the mAb were renewed accordingly.The culture medium to be discarded was checked to make sure that no eggs were lost. The cultures were generally kept for 6 days until the worm pairs began to dissociate.The eggs produced were counted each day under phase contrast microscopy. The assay was duplicated in each group.

about 69 f 1%of the original enzymatic activity,while with 100 pl and 200 pl of SN, only 51 f 2% and 37 k 3% of GST activity remained. Corresponding doses of the control C3 mAb SN produced no inhibition (Fig. 1A). The dosedependent inhibitory effect was confirmed with ascitesfluid and purified IgM as demonstrated in Fig. 1B and C. The blocking effect of S13 mAb on Sm 28 GSTactivity was shown to be specific for the following reasons. First, as mentioned above, although H12 mAb can recognize Sm 28 GST, it did not inhibit the enzymatic activity (Fig. 1B). Secondly, S13 mAb showed no inhibition of rat liver GST, A

In the ex vivo model, adult worm pairs recovered from S13or control mAb-passivelytransfered mice were cultivated in the same way except that there was no addition of mAb ascites fluids.

"

I

0

50

100

150

200

Supernatant

(PI)

2.8 Egg hatching assay B

Schistosome eggs were collected from hamsters infected with S. mansoni 45 days previously [17]. Briefly, the livers were homogenized on ice and the cell preparations were washed several times by centrifugation (4OOO rpm at 4 "C in the dark for 15 min). About 100 eggs were incubated with S13, H12 or IA ascites fluids (100, 200 and 500 pl) in a 6-well plate (Falcon, Oxnard, CA) at 4°C for 4 h before stimulating egg hatching at 30°C in full light for 1h, the volume of each well being adjusted to 10 ml with calciumfree water. The hatching was stopped with 100 pl 1% (v/v) Lugol solution.The miracidia and living and non-living eggs were then counted under a phase contrast microscope.

"

I

0

10

20

40

30

Ascites

(PI)

The hatching assay was also performed using eggs recovered from the small intestines of the mAb-treated mice as described above, except that no preincubation with Ab was carried out in this case. 2.9 Statistics

The data were presented as means k SE.The significanceof the results were calculated by Student's t-test.

ee 8

20-

01

3 Results 3.1 Dose-dependent inhibition of the expression of Sm 28 GST activity by SW mAb

After incubation with three different preparations of S13 mAb, cell culture SN, ascites fluid or the purified IgM fraction, the Sm 28 GST activity was inhibited in a dose-dependent manner (Fig, 1). In the linear part of the curve, Sm 28 GST incubated with 50 pl of a tenfold concentrated serum-free S13 cell culture SN exhibited

1803

g g3 1

1804

mol

Eur. J. Immunol. 1991.21: 1801-1807

C-R. Xu, CVerwaerde, J.-M. Graych et al.

when the incubation was performed in the same way as for Sm 28 GST (data not shown).

T

3.2 Worm burden reduetion by mAb transfer The in vivo effects of Sm 28 GST blocking mAb on schistosome infection was studied by passive transfer of the mAb to rats and mice. The first parameter to indicate a protective effect was the reduction in worm burden. Rats that received anti-schistomme Sm 28 GST mAb, S13 and H12, developed a significant resistance to the challenge infection of 45 and 34%, respectively,when compared to the IgM control mAb (IA) recipients (Fig. 2). IA

H12

S13

Ascites

Figure 2. Worm burden in rats passively transfered with anti-Sm28 GSTmAb. Eight male Fischer rats of each group were infected with lo00 S. mansoni cercariae and i.v. injected with 2 ml IgM control mAb IA or anti-Sm 28 GST mAb, S13 or H12 ascites fluids 4 h 1ater.The liver perfusion was performed 20 days post-infection and the recovered worms were counted. The worm burden reduction was calculated according to the formula given in Sect. 2 using IA-treated group as control. The significanceof reduction wasp < 0.001 for S13 group and p < 0.005 for HI2 group.

Worm burden reduction in mice that received anti-schistosome Sm 28 GST mAb was generally weak (0 to 28.6%, Table 1). However, the diminution in worm recovery was significant when the mAb was transfered i.v. 3.3 Impairment of worm fecundity by the Sm 28 GST blocking mAb

Although the effects of anti-Sm 28 GST mAb on worm burdens in mice were not highly significant, the effects on

Table 1. Worm recovery in mice passively transfered with mAbs Timing” Triinsfcr Ascitcs

DO

i.p. i .v. i.p. i .v.

030

i.p.

4 6 4 8

45.8 f 13.6 19.4 f 14.6 55.7 f 9.4 45.8 +- 8.5

-

-

0

-

NSd)

27.8

0.035

1A S13 IA S13

n

41.1 f 13.9 41.5 f 11.2

0

47.8 k 5.8 34.4 k 8.0

-

NS -

28.0

0.032

IA H12

7 7 7

IA

7 6 8

IR S13 IR

s1.7

i .v.

p

Worm burdenb)

S13

DO+D 1.5

% Reduction‘)

Animal numbers

HI2 s13

8 8 8

57.2 k 57.0 f 57.4 f 62.8 2

14.0 6.5 15.5 10.4

4.8 f 12.3 47.1 f 16.8

-

-

-

0 0

NS

-

NS

28.6 25.0

0.045 0.045

-

a) BALB/c mice were infected with 100 S. mansoni cercariae and injected either i.p. or i.v. with IR or IA, H12 or S13 mAb preparations at different times post-infection : day 0 at 4 h, day 15 or day 30. b) Whole body perfusion was carried out 45 to 48 days after infection and worms were counted. c) IR and IA were used as controls in different experiments. d) “NS” means not significant.

Table 2. Effects of mAb passive transfer on egg production and egg viability in mice % Increase of non-living eggsh)

% Reductionh)

Timing“) Transfer

Ascites Intestinal eggs

Do DO+D15

D31)

.

Hatching

S13 S13

7.1 28.9

NS

I .v.

*

36.1 42.8

i.p.

S13

I .v.

S13

42.7 31.0

** **

63.6 49.0

i.p.

HI2

NS

i .v.

S13 HI2 S13

3.3 40.9 10.2 47.2

i,. p

16.0 60.0 27.4 50.5

*** * ***

* ** *** *** NS

*** NS

***

13.8 0

NS

47.6 45.8

** **

0 43.9 8.9 30.9

*** NS

**

Experiments corresponding to those described in ’Ihble 1. b)Percentage of reduction or increase was calculated by comparison to IA-transfered group. The signs “*”, “**” and “***” denot. p < 0.05, p < 0.01 andp < 0.001, respectively, “NS”means not significant.

Eur. J. Immunol. 1991. 21: 1801-1807

Immune effects of anti-Sm 28 GST mAb in S. mansoni

The inhibitory effects of S13 mAb on adult schistosome fecundity were also observed ex vivo after the culture of worm pairs recovered from in vivo mAb passively transfered mice (Fig. 3). The worm pairs from S13-treated mice laid markedly fewer eggs than their counterparts receiving IA and H12. During the first 4 days of in vitro culture, egg laying was decreased by 23 to 43%.

a

Ul Ul

al

c 0 L

Q

n

f

2

2

4

3

Days

Figure 3. Egg-laying impairment in an ex vivo model. Adult worm pairs were recovered from mice that were infected with 100 S. mansoni cercariae and passively transfered with either IgM control mAb IA, or anti8m 28 GSTmAb S13 or H12. Egg production was followed in in vitro cultures in RPMI 1640 medium supplemented M 2-ME and 108sheep with 20% FCS, 0.1% of glucose and 5 X erythrocytes/ml. Eggs were counted each day up to day 4 when the worm couples began to dissociate. This graph illustrates one representative experiment in which data were collected from each three pairs of worms recovered from 2 mice in each group.

schistosome egg deposition in the small intestines were clear.The deposition of eggs in mice that received S13 mAb was reduced by about 30 to 60% (Table 2) even in the absence of a reduction in parasite load in some of the experiments. On the other hand, no reduced egg deposition was noticed in HlZtransfered mice. A

c

a

Ul 600 0 Q c 9 0 0 L

0

f”

8

200

:

loo o

: 0

,

,

,

I

1

2

3

4

,

1

5

6

3.4 Impairment of egg viability by Sm 28 GST blocking mAb

Hatching assays performed using schistosome eggs recovered from mice that had previously received S13 mAb revealed that egg viability was markedly affected whatever the timing of the passive transfer (Table 2). H12 mAb showed no such effects. In addition to the inhibition of egg development into miracidia, the transfer of S13 mAb also significantly increased the proportion of non-living eggs (“brown”eggs and “black” eggs; Table 2). The “brown”eggs correspond to immature eggs containing a mass of nondifferentiated cellinside the egg shell while the “b1ack”eggs contain a dead mature miracidium [18].

Table3. Effects of S13 mAb on schistosome egg hatching in vitro

Days 6oo

These effects on egg production were further confirmed by in v i m experiments.The adult worm pairs recovered from hamsters previously infected with S. rnansoni produced significantly fewer eggs when incubated in vitro in the presence of S13mAb than those incubated with the anti-Sm 28 GSTcontrolmAb, H12, or the IgM isotype control mAb, IA (Fig. 4). The blocking activity of S13 on egg laying was dose dependent with 100 pl of ascites fluid exerting an average of 30% inhibition and 200~1,51% (Fig. 4A). Similar results were obtained with the purified S13 IgM fraction (25 to 66% of inhibition) (Fig. 4B).

These observations on in vivo treated eggs were confirmed by the in vitro incubation of normal schistosome eggs in the presence of the Sm 28 GST-blockingmAb S13 (Table 3). A marked significant reduction in egg hatching was produced by S13 although the effect was not dose dependent in the second experiment. In two separate experiments H12 failed

400

n

=

B

1

% 0

% Hatching reductionh)

Ekpt. No. 1

mAb Sources”)

al

S13

loo@

60.5

uw)pl

70.7 83.5

mpl

H12

100 fl uw)

-

1805

fl

-4

0 0

1

2

3

4

5

5.0 33.9 0

Expt. No. 2

*** *** ***

56.4

54.4

** ** ***

NS

13.1 16.5

NS NS

*

61.3

0

6

Days

Figure 4. The impairing effects of S13 mAb upon schistosome egg production in vitro. Three pairs of adult worms recovered from 45-day-infected hamsters were cultured in vitro as described in Fig. 3 in the presence of different mAb preparatians. (A) C3,H12 and S13ascitesfluids. (B) C3 and S13purified 1gM.The data in the graph represent the mean and SE of three experiments.

a) Schistosomeeggs collected from 45 day-infected hamsters were incubated in vitro for 5 h with different doses of anti-Sm 28 GST mAb S13 or H12. An irrelevant IgM isotype IA mAb was employed as control. b) Percentage of hatching reduction was calculated using IA treated-group as control.The meanings of “*”, “**”, “***”and “NS”were p < 0.05, p < 0.01, p < 0.001 and not significant, respectively.

1806

C-B. Xu, C.Verwaerde, J.-M. Grzych et al.

Eur. J. Immunol. 1991.21: 1801-1807

pared to that seen in rats) was consistent with the previous and failed attempt to transfer protection in mice with mouse anti-Sm 28 GST sera [32]. It is generally suspected that the immunity elicited in murine schistosomiasis either by chronic infection or by various methods of vaccination 4 Discussion mainly relies on cell-mediated mechanisms rather than on An S. mansoni 28-kDa Ag has been shown to be protective the humoral response [33]. in various animal models [191. Previous work has generally been focused on the humoral and cellular immune response In spite of the less evident and variable worm burden [7].The demonstrationof the GST (EC 2.5.1.18) activity of reduction, a significantly reduced egg deposition was this Ag [3] allowed us to put forward the hypothesis that the noticed in the intestines of mice passively transfered with inhibition of the GST-activity might constitute an alterna- the Sm 28 GST inhibitory mAb (S13) but not with the GST tive approach to explain the protective effects observed in non-inhibitory mAb (H12). In addition, the hatching of Sm 28 GST immunization, as previously proposed for other these eggs into miracidia was severely impaired (Table 2). schistosome GST by G. Mitchell [20]. This idea was based The reduced egg production was confirmed by in ex vivo on the accumulating chemical and biological evidence studies (Fig. 3) and in in vitro cultureof normal adult worms indicating that GST-catalyzed mechanisms using reduced in the presence of S13 mAb (Fig. 4). The S13 mAb also GSH play a vital role in detoxification in a wide range of showed an inhibitory effect on egg hatching in in vitro animal species. Earlier reports suggest that GSH may have incubation (Table 3). In support of our observations, a an essential role in schistosome detoxification and resis- diminution in egg production has also been noticed recently tance mechanisms against chemotherapeuticdrugs, includ- after active vaccination with Sm 28 GST [34]. Reduced egg ing niridazole and oltipraz [21,22]. GSH is also well known production was noted in Sm 28 GST-immunized baboons to participate directly or indirectly in many vital biochem- [35] and impaired egg hatching was noted when a combiical process, including the synthesis of protein and DNA, nation of immunization with Sm 28 GSTand praziquantel transportation, enzyme activity, sugar and lipid metabolic treatment was employed in mice (Grezel, D., personal pathways, and the protection of cells [23]. In schisto- communication). somiasis studies, the reported influence of schistosome worm pairing on the level of GSH in male [24,25] and the The mechanism responsible for these effects remains to be regulation of a schistosome hemoglobin-degrading pro- explored. For the in vivo model, the lifespan of rat mAb in tease by GSH activation [26], suggest the importance of this the mouse circulation (no data available) should be considmolecule in the metabolism of the parasite. In the present ered.The half-lives of homologous IgM was 25 h in rats [36], work, we show for the first time that inhibition of Sm 28 1day in mice and 5 days in humans [37]. The timing (see GST can cause damage to some of the essential life Table 1) of transfer of IgM mAb in mice reflected the functions of the parasite, i.e. egg production and viabili- considerations on the clearance of IgM from the circulation. ty.

to show any significant reduction in egg hatching at the same Ab concentrations.

An mAb, S13, was selected for its ability to block the GST activity of Sm 28 GST. Another anti-Sm 28 GSTmAb, H12, of the same isotype (IgM), but lacking the GST inhibitory activity,was used as a control.The inhibition of Sm 28 GST by the S13 mAb was dose dependent but not linear, as is the case for the inhibition of various GST by many kinds of inhibitors, including heavy metals [27, 281, plant phenols [29], and biologicallyactive materials, such as bile acid [30]. This inhibitory action seems to be specific to the S13 mAb since H12 showed no effect. Moreover, S13 had no inhibitory effect on rat liver GST. To study the relationship of the blockade of GST activity and the deduced effects on schistosome infection, the anti-Sm 28 GST mAb were passively transfered into rats and mice. As far as worm burden reduction was concerned, the protection transfered was similar to that seen upon active immunization with Sm 28 GST [4, 311 and upon passive transfer of Sm 28 GST-specific T h cells [31, 321 in rats. Of the two mAb, S13 (which blocks Sm 28 GST activity) did not give a Significantly higher reduction in worm burden than did H12 (which does not inhibit GST activity) when the same quantities of Ab were transfered (Fig. 2) suggesting that Sm 28 GST blockade is not necessary for worm elimination. In mice, the worm burden was at best only slightlyreduced. Significant reductions only occured when Ab transfer was effected i.v. (Table 1). This less marked diminution (com-

As a protective Ag provoking host immune responses and as a GST enzyme involved in many crucial parasite metabolic pathways, Sm 28 GST plays a dual role in the parasite-host interplay.To explain the persistent effects of the mAb on worm egg production when a single early transfer (day 0) was performed, the influence of the mAb upon schistosomesurvival and maturation seems to be the major cause. This hypothesis was supported first by the parallel between the decreases in worm burden and egg deposition : a significant reduction in egg numbers was observed when significantly less worms were recovered (Tables 1and 2, i.v. transfer). Secondly, a slightly increased number of immature worms was obtained in S13 mAbtransfered mice (data not shown). However, one later injection or two injections of S13 mAb resulted in a similarly reduced egg deposition in the small intestines that was independent of the worm burden.These effects were rather due to the blockade of the Sm 28 GST activity since the non-blockingmAb, H12, showed no effect on egg production even though a slight reduction in worm numbers was obtained (Thbles 1 and 2). This direct influence of S13 mAb on the worm metabolism was reproduced in vitro. Hepatic granuloma formation elicited by the Ag released from eggs entrapped in the host liver is the major manifestation of schistosomiasis pathology [38, 391. Indeed, it is generally believed that the T lymphocyte-mediatedDTH is

Eur. J. Immunol. 1991. 21: 1801-1807

Immune effects of anti-Sm 28 GST mAb in S. mansoni

the principal initiator and that the mature eggs containing miracidia are the sources of immunopathogenic Ag in S. mansoni [40]. Sm 28 GST is expressed in different developmental stages of the parasite, maintaining a constant stimulation to elicit a permanent anti-Sm 28 GST response. It can be expected that the various effects of the GST-blocking Ab on parasite elimination,worm fecundity and egg viability act in an additive fashion.

10 Lowry, 0.H., Rosebrough, N. J-, Farr, A. L. and Randall, R. J., J. Biol. Chem. 1951. 193: 265. 11 Grzych, J.-M., Capron, M., Bazin, H. and Capron, A., J. Immunol. 1982.129: 2739. 12 Des Moutis, I., Ouaissi, A., Grzych, J.’ M., Yarzabal, L., Haque, A. and Capron, A., Am. J. Trop. Med. Hyg. 1983.32: 533. 13 Bazin, H., Beckers, A. and Querinjean, I?, Eur. J. Immunol. 1974. 4: 44. 14 Habig, W. H., Pabst, M. J. and Jacoby, W. B., J. Biol. Chem. 1974.249: 7310. 15 Smithers, S. R. and Rrry, R. J., Parasitology 1965. 55: 695. 16 Doughty, B. L. and Phillips, S. M., J. Immunol. 1982. 128: 30. 17 Nirde, I?,’Ibrpier, G., De Re@, M. L. and Capron, A., FEBS Letters. 1983. 151: 223. 18 Pellegrino, J. and Katz, N., Adv. Parasitol. 1968. 6: 233. 19 Capron, A., Pierce, R., Balloul, J. M., Grzych, J. M., Dissous, C., Sondermeyer, I? and Lecocq, J. I?, Acta Tropica 1987. 44 (Suppl. 12): 63. 20 Mitchell, G. F., Parasitol. Today 1989. 5: 34. 21 Tracy, J. W., Catto, B. A. and Webster, L. T., Mol. Pharmacol. 1983.24: 291. 22 Morrison, D. D.,Thompson, D. F!, Semeyn, D. R. andBennett, J. L., Biochem. P h a m c o l . 1987. 36: 1169. 23 Meister, A. and Anderson, M. E., Annu. Rev. Biochem. 1983. 52: 711. 24 Siegel, D. A. and Tracy, J. W., J. Parasitol. 1988. 74: 524. 25 Siegel, D. A. and Tracy, J. W., Exp. Parasitol. 1989. 69: 116. 26 Chappell, C. L., Dresden, M. H. and Walters, D.W., Biochem. Bwphys. Acta 1987. 913: 335. 27 Dierickx, F! J., Pharmacol. Res. Commun. 1985. 17: 489. 28 Dierickx, F! J., Enzyme 1982. 27: 25. 29 Das, M., Bickers, D. R. and Mukhtar, H., Biochem. Biophys. Res. Commun. 1984.120: 427. 30 Hayes, J. D. and Mantle,T. J., Biochem. J. 1986. 233: 407. 31 Auriault, C., Balloul, J. M., Pierce, R. J., Damonneville, M., Sondermeyer, F! and Capron, A., Infect. Immun. 1987. 55: 1163. 32 Wolowczuk, I., Auriault, C., Gras-Masse, H. ,Vendeville, C., Balloul, J.-M., Tartar, A. and Capron, A., J. Immunol. 1989. 142: 1342. 33 James, S. L. and Sher, A. in Kaufmann, S. H. E. K(Ed.), T-cell Paradigms in Parasitic and Bacterial Infections. CTMI 155. Springer-Verlag, Berlin 1990, p. 21. 34 Capron, A., Balloul, J. M., Grezel, D., Grzych, J. M., Wolowczuk, I., Auriault, C., Boulanger, D., Capron, M. and Pierce, R. J., in Melchers, F., et al. (Eds.), Progress in Irnmunologx Vol.VIZ, Springer-Verlag, Berlin and Heidelberg 1989, p. 979. 35 Boulanger, D. Parasite Zmmunol. 1991, in press. 36 Peppard, J.V. and Orlans, E., Immunology 1980. 40: 683. 37 Spiegelberg, H. L., Adv. Immunol. 1974. 19: 259. 38 Hang, L. M.,Warren, K. S. and Boros, D. L., Exp. Parasitol. 1974. 35: 288. 39 Warren, K. S., Zmmunol. Rev. 1982. 61: 189. 40 Garb, K. S., Stavisky, A. B., Olds, G. R., Tracy, J. W. and Mahmoud, A. A. F., J. Immunol. 1982. 129: 2752.

Taken together, these observationssuggest that the study of a potential vaccine Ag should not only focus on the definition of the immunodominant B and T epitopes, but should equally take into account the possible effect of the immune response on the expression of the biological function of the Ag. Our report supports the view that schistosome GST antigens may act not only as an active vaccine candidatebut also in limiting the parasite transmission and in alleviating pathological manifestations by its effects on the eggs. We would like to thank Dr. Monique Capron, Dr. Jean-Claude Ameisen, Dr. Raymond J. Pierce, Dr. Isabelle Wolowczuk, Delphine Grezel and Denis Boulangerfor their helpful discussions, Ms. Anne-Marie Schacht and Sophia Lafittefor their technical help, and Mrs. Monique Leroun for providing S. mansoni life cycle materials. Received March 25. 1991.

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