Surface antigens on cercariae, schistosomula and adult worms of Schistosoma mansoni

OOZO-7519/82/050451-11603.00/0 Pergamon Press Ltd. Society for Parasitology

( 1982 Ausfrolron

SURFACE

ANTIGENS ON CERCARIAE, SCHISTOSOMULA ADULT WORMS OF SCHISTOSOMA MANSONI J. SHAH*

Department

of Biochemistry,

AND

and R. RAMASAMYj-

University

of Nairobi,

P.O.

Box 30197, Nairobi,

Kenya

(Received 26 June 198 1) Abstract-SH.u J. and RAMASAMY R. 1982. Surface antigens on cercariae, schistosomula and adult worms of Schistosoma mansoni. International Journal for Parasitology 12: 451-461. The surface protein antigens of Schistosoma mansoni were radiolabelled by lactoperoxidase catalysed 1r2s-iodination and analysed by immune-precipitation and polyacrylamide gel electrophoresis. The results showed that regularly labelled surface antigens of mol. wts >150,000, 78,000, 45,000 and 22,000 were present on adult worms. Common surface antigens were observed on the cercariae, schistosomula and adult worms. It is suggested that surface antigens released from living adult worms can sensitise a host to react against the invading schistosomula of a secondary infection. However, the failure to vaccinate mice using material containing adult worm surface antigens suggests that the induction of protective immunity is a complex phenomenon.

INDEX KEY WORDS: Adult worms; Bolton-Hunter reagent; cercariae; phoresis; immunity to parasites; lactoperoxidase; membrane turnover; mansoni; schistosomiasis; schistosomula; surface antigens; surface trematode.

INTRODUCTION

concomitant immunity; electroparasite surface; Schistosoma iodination; surface proteins;

1973). penetrates the skin (Hockley & McLaren, Adult S. mansoni worms are resistant to an immune response mounted by the host and can survive for several years in the mesenteric veins. The mechanisms of this immune evasion are not clearly understood. Possible escape mechanisms include the development of the double outer membrane (Hockley & McLaren, 1973), the rapid turnover of the outer membrane components (Perez & Terry, 1973) and the lowering of parasite immunogenicity by the acquisition of a coat of host antigens (Smithers, Terry & Hockley, 1969). While the adult worms are resistant to a host immune response, the invading schistosomula of a secondary infection are rapidly destroyed by the host. This phenomenon has been termed concomitant immunity (Smithers & Terry, 1969). A number of immunological mechanisms that result in the death or severe damage of schistosomula in vitro, involving the action of antibody and complement (Clegg & Smithers, 1972) or antibodies and cells (Butterworth, Sturrock, Houba, Mahmoud, Sher & Rees, 1975; Capron, Bazin, Desaint & Capron, 1975; Perez & Smithers, 1977), have been described. A central feature in these mechanisms is the presence of molecules on the surface of the schistosomulum that are antigenic in the host. The surface antigens of the adult worm and the schistosomulum are therefore clearly important in understanding the interaction between the host immune system and the parasite. The detection of surface protein antigens on adult worms after surface

HUMAN schistosomiasis is a major tropical parasitic disease. Schistosoma mansoni, which causes intestinal schistosomiasis, is prevalent in many African countries, parts of the Arabian peninsula, in South America and the Caribbean Islands. The infective form of the parasite causing schistosomiasis is the cercaria released by the intermediate snail host. During the process of infection, the cercariae which penetrate the skin of the mammalian host are transformed into immature worms called schistosomula. Schistosomula then mature into adult worms that live in the mesenteric veins of the host. Changes in the parasite tegument take place during the transformation of the cercaria into a schistosomulum. These changes may be relevant to the evasion of a host immune response by the parasite. The parasite tegument is a syncitium and in the cercaria is bounded on the outer surface by a unit (trilaminate) membrane. In the schistosomulum and the adult worm, the outer membrane of the tegument is made up of two closely apposed unit membranes which gives a heptalaminate appearance under the electron microscope. The formation of the double membrane is essentially complete 3 h after the cercaria *Present address: ILRAD, P.O. Box 30709, Nairobi, Kenya. j-Correspondence: R. Ramasamy, Department of Biochemistry, Faculty of Medicine, University of Jaffna, Jaffna, Sri Lanka. 451

.I. SHAH and R. RAMASAMY

452

1125 radiolabelling has been reported (Kusel, Mackenzie & McLaren, 1975, Hayunga, Murrel, Taylor & Vannier, 1979a & b). The surface proteins of cercariae and schistosomula of S. mansoni have also been labelled with 112s (Ramasamy, 1979). The work reported here is the characterisation of surface antigens on cercariae, schistosomula and adult worms of S. mansoni after surface 1125labelling of the parasite. A preliminary evaluation of the use of surface antigens for protective immunisation against schistosomiasis is also described. MATERIALS AND METHODS Parasites. A strain of S. mansoni originating in the vicinity of Nairobi, Kenya, was used throughout these experiments. Adult worms were obtained by perfusing CBA mice that had been infected percutaneously six weeks previously with cercariae. The mice were killed by injecting heparinised Nembutal intraperitoneally. The hepatic portal vein was then cut and the mice perfused with titrated saline (0.15 M sodium chloride and the 0.05 M sodium citrate) to release adult worms. The worms were collected on a sieve, washed three times in phosphate buffered saline, pH 7.2 (PBS) and counted. The worms were used as soon as possible, generally within 1 h after isolation, for radiolabelling experiments. The preparations used in the experiments contained approximately equal numbers of male and female worms. Cercariae were collected from laboratory infected snails by exposing them to light in a beaker of water. The cercariae were cooled to 4”C, to reduce their mobility and then washed by centrifugation in PBS. Schistosomula were produced from cercariae by the skin penetration technique using mouse skins (Clegg & Smithers, 1972). Schistosomula were collected 3 h after the cercariae were allowed into contact with the skins. The schistosomula were then washed three times in PBS by centrifugation. Only preparations of schistosomula that were contaminated with less than 5% cercariae were used in the experiments. Rabbit antisera and human sera. A rabbit antiserum was raised against adult worm freeze thaw antigens (AFT) obtained by rapidly freezing and thawing worms in PBS three times as described by Kusel (1972). Electrophoretic analysis showed that such preparations contained soluble proteins released by the worms as well as worm teguments. 0.5 ml of AFT from 250 worms in PBS (approximately 0.25 mg of protein), was emulsified with 0.5 ml Freund’s complete adjuvant and injected sub-cutaneously into a rabbit. The rabbit was then injected fortnightly with the same amount of AFT in PBS and bled 10 days after the third injection. Thereafter a booster injection was given in PBS, 10 days before bleeding the rabbit. Rabbit antisera to ovalbumin, lactoperoxidase (Sigma, England), whole mouse serum and human IgG (prepared in our laboratory and shown to be pure by immunoelectrophoresis) were raised in a similar manner. Sera (S7 and S12) were also obtained from patients carrying active S. mansoni infections identified at the Kenyatta National Hospital, Nairobi. The clinical picture of infection was confirmed by the detection of eggs in the stools. Surface radiolabelling with I12’. The parasites were radiolabelled by lactoperoxidase catalysed iodination using a modification of the method described by Marchalonis, Cone & Santer (1971). This technique results in the incorporation of 1t25 into unsaturated lipids and exposed tyrosine and to a lesser extent histidine, residues of proteins

1.1.~. VOL. 12. 1982

present on the outside of the plasma membranes of intact cells. This is because the active iodinaring species is formed near the enzyme which, being a large protein, does not penetrate an intact plasma membrane (Phillips & Morrison, 1970). In our experiments, 100-200 adult worms, 15,00020,000 schistosomula or 15,000-20,000 cercariae in 0.5 ml PBS were placed in a conical ended plastic tube at room temperature. In each was added, in sequence, 0.03 mg lactoperoxidase in PBS and 37 MBq of Na1t2s (Radiochemical Centre, England). The reaction was initiated by the addition of 0.01 ml of 0.03% w/v hydrogen peroxide and allowed to proceed for 10 min. Further additions of 0.01 ml of 0.03% hydrogen peroxide were made 3 and 6 min after the beginning of the reaction. The parasites were gently mixed at frequent intervals during the labelling procedure. The reaction was terminated by the addition of 10 ml of PBS. The parasites were then washed three times by centrifugation in 10 ml PBS and transferred to fresh tubes for extraction of radioiabelled proteins. The parasites were not killed or seriously damaged by the labelling procedure. This was shown by the retention of parasite mobility at the end of the labelling. Adult worms and schistosomula were also labelled with the Bolton-Hunter reagent, lt2s-iodinated Nsuccinimidyl p-hydroxyphenylpropionate, exactly as described by Hayunga et al. (1979a) except for the numbers of parasites. 150-200 adult worms and 15,000-20,000 schistosomula were used in our experiments. Surface glycoproteins on the parasites were labelled with HJ by the method of Gahmberg and Hakamori (1973). Extraction of labeled proteins. For the analysis of labelled surface proteins by electrophoresis, 150-200 worms and 15,000-20,000 schistosomula or cercariae were treated with 0.2 ml 1% sodium dodecyl sulphate (SDS) for 15 min at room temperature. The insoluble parasite material was then removed by centrifugation at 2000 g for 5 min. Before electrophoresis, 0.2 ml of the SDS extract was treated with 0.1 ml of sample dissolving solution (Laemmli, 1971) containing 0.06 M Tris-HCl buffer pH 6.3, 2% SDS, 10% glycerol, 5% mercaptoethanol and 0.01% bromophenol blue and placed in a boiling waterbath for 2 min. For immune-precipitation of labelled surface proteins, 500 worms and 20,000 schistosomula or cercariae were routinely extracted in 1 ml of 1% sodium cholate pH ‘.2 containing I mM phenylmethylsulphonylfluoride (PMSF), a protease inhibitor. The extraction was carried out for 15 min at room temperature. In certain experiments the extraction was performed using 1% Nonidet P40 (Fluka, Switzerland) in place of 1% sodium cholate. Nonidet P40 is a weak non-ionic detergent while sodium cholate is a weak ionic detergent. Insoluble parasite material was removed from the detergent extracts by centrifugation at 2000 g for 5 min. Release of radiolabelled material from worms. Washed 1125 labelled adult worms were incubated at a concentration of 50 worms/ml in Hank’s medium and PBS at room temperature. The rate of release of radioactivity was followed by removing 20 ~1 aliquots of the medium in triplicate at different time intervals. The remaining medium was collected after 24 h and aliquots used for immune-precipitation with rabbit anti-AFT and rabbit anti-ovalbumin sera. Immune-precipifation. 0.1 ml of the cholate extract of the parasites was equivalent to 50 adult worms, 2ooO schistosomula or 2ooO cercariae. For direct immune-precipitations, 0.1 ml of the extract was allowed to react with 0.4 ml of the rabbit anti-AFT serum for 16 h at 4°C. Carrier unlabelled AFT equivalent to 50 worms was added in the case of schis-

I.J.P. VOL. 12. 1982

Surface

antigens

tosomula and cercariae. Control direct immune-precipitates were formed by the addition of 0.4 ml of the rabbit antisera to ovalbumin and 0.05 mg of ovalbumin to 0.1 ml aliquots of parasite extracts. For indirect immune-precipitations with the human sera, 0.1 ml of the extract was allowed to react with 0.02 ml of the serum for 16 h at 4°C. The antigen-antibody complexes were then precipitated by the addition of 1 ml of the rabbit anti-human IgG serum and incubation for 2 h at 37°C. The immune-precipitates were centrifuged to a pellet at 2000 g for 5 min at room temperature and then washed once in 1% cholate (to remove nonspecifically adherent membrane material) and twice in PBS. The precipitates were then treated with 0.2 ml sample dissolving solution at 37°C for 1 h and then placed in a boiling waterbath for 2 min to complete the dissolution.

Detection

453

on Schistosoma mansoni

of lactoperoxidasein labelledparasite extracts.

0.1 ml aliquots of parasite extracts were reacted with 0.02 ml of rabbit anti-lactoperoxidase serum and 0.02 ml of rabbit anti-ovalbumin serum as a control for 6 h at 4°C. 0.2 ml of a 10% suspension of protein A-sepharose (Pharmacia, Sweden) was then added to each reaction mixture to bind rabbit IgG (Kessler, 1975) for a further period of 10 h at 4°C. The beads were then washed three times and the bound labelled proteins extracted in 0.2 ml sample dissolving solution by heating in a boiling water bath for 2 min. Electrophoresis and autoradiography. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out on vertical slab gels according to the method of Laemmh (1971). Electrophoresis was performed under reducing conditions on 10% gels. Kodirex or NS-2T X-ray films (Kodak, England) were used for autoradiography of dried 1’25 labelled gels while X-omat film was used for H3 autoradiography according to the method of Bonner & Laskey (1974). Details of the electrophoretic procedure, autoradiography and the determination of the mol. wts of labelled proteins have been described elsewhere (Ramasamy, Jamnadas & Mutinga, 1981). Electron microscopy of worms. Adult worms were shamlabelled with non-radioactive sodium iodide using conditions identical to the real procedure. The worms were fixed in glutaraldehyde, post fixed in osmium tetroxide; washed and embedded in Epikote. The blocks were sectioned using a Sorvall Ultra microtome MT-l and mounted on copper grids. The sections were then stained with many1 acetate and lead citrate and examined in an AEI Corinth 275 electron microscope. Autoradi0graph.v of labelled worms. Autoradiography of labelled worms was performed at the International Laboratory for Research on Animal Diseases, Nairobi. The worms were washed after I t2s labelling, fixed in Bouin’s solution, embedded in paraffin wax and sectioned. The sections were transferred to glass slides, fixed and autoradiography carried out according to the method of Oaks & Lumsden (1971). The sections were stained with haemotoxylin and eosin after development to enhance contrast. Immunofluorescence. The reaction of antibodies with surface antigens on cercariae, schistosomula and adult worms was monitored by indirect immunofluorescence. Washed, freshly isolated parasites (IO worms or 50 schistosomula and cercariae) were reacted with mouse, rabbit or human sera at different dilutions (0.05 ml) for 30 min at 4°C. The parasites were then washed three times in PBS and treated with an optimal dilution of fluorescein conjugated IgG fraction of the appropriate anti-IgG serum for 30 min at 4°C. ‘The parasites were washed three times in PBS and viewed under U.V. illumination in a Leitr fluor-

escence microscope. Surface sites was scored as a positive

fluorescence reaction.

on motile

para-

Immune-protection experiments. Age and sex matched groups of CBA mice were injected intra-peritoneally with AFT in PBS or emulsified in Freund’s complete adjuvant. AFT derived from 50 worms were used for a single injection. The injections were repeated at 10 day intervals with AFT in PBS. After two injections in PBS, the sera from the mice were monitored for antibodies to the surfaces of schistosomula by immunofluorescence staining of freshly prepared, viable schistosomula. The sera of control groups of mice injected with Freund’s complete adjuvant followed by PBS or PBS only were also examined. After the course of injections described here, mice injected with AFT, but not the control groups of mice, were found to have antibodies to surface antigens of schistosomula. The four groups of mice were then infected with 400 cercariae percutaneously and the adult worms obtained by perfusion and counted six weeks after the infection.

RESULTS

Surface proteins in S. mansoni The Coomassie blue staining patterns of SDS extracts of Ii*5 labelled cercaria, schistosomula and adult worms, analysed by SDS-PAGE, were similar and showed that the procedure resolved proteins in the mol. wt. ran:e from 15,000 to more than 100,000. The similalrty in the composition of the major parasite proteins has been noted previously (Ruppel & Cioli, 1977). In the presence of SDS and mercaptoethanol, polymeric proteins are dissociated into subunits and the protein composition observed in the gels reflects the composition of individual polypeptide chains. Autoradiographs obtained from the radioactive gels revealed a number of proteins that were labelled with 1125 by lactoperoxidase catalysed iodination (Fig. 1). A prominently labelled 76,000 mol. wt. protein was detected in all three forms of the parasite. The background of radioactivity in gels of SDS extracts was rather high and this did not readily permit the detection of many of the weakly labelled proteins. A comparison of lactoperoxidase catalysed iodination with iodination by the Bolton-Hunter reagent revealed that the prominent 76,000 mol. wt. protein was absent in extracts of Bolton-Hunter reagent labelled parasites. The patterns of labelled proteins produced by the two techniques from either schistosomula or adult worms were not identical. However several common proteins were labelled by the two techniques. The radiolabelling technique of Gahmberg & Hakamori (1973) for detecting surface glycoproteins on cells did not prove very satisfactory for schistosomes. Several labelled bands were observed but many of these were also present when galactose oxidase was omitted in control labelling procedures. A high mol. wt. glycoprotein (>150,000 mol. wt.) was however specifically labelled on adult worms. A glycoprotein migrating in a similar position on gels was also observed when AFT was analysed by SDS-

J. SHAH and R. RAMASAMY

454

‘3

Abh 3

‘bh

I.I.P. VOL. 12. 1982

‘1

-130K

-43K -34.7K

-24K FIG. 2. Electron micrograph showing the intact tegument of an adult worm sham-labelled by lactoperoxidase catalysed iodination.

Surface antigens on adult worms

FIG. 1. 112s labelled

proteins

of S.

mansoni extracted

in

SDS. C,, S, and A, are extracts of cercariae, schistosomula and adult worms respectively labelled by lactoperoxidase catalysed iodination. Sbh and A,, are extracts of schistosomula and adult worms labelled with the Bolton-Hunter reagent.

Migration positions of marker proteins phoresed in parallel tracks are indicated.

PAGE and the gels stained for carbohydrate Periodic acid-Schiff reaction.

electro-

by the

Surface localisation of proteins Iabelled by the lactoperoxidase technique The lactoperoxidase labelling technique was selected for further experiments because of the simplicity of the procedure, the efficiency of labelling and the relative inexpensiveness of the labelling reagent. However before carrying out detailed investigations, it was felt desirable to determine whether the parasite tegument was damaged by the labelling procedure and whether the labelled proteins were localised on the surface of the parasite. Electron microscopic observations of sham-labelled adult worms showed that the parasite tegument was intact and not damaged by the conditions used for labelling (Fig. 2). Autoradiography of sections of labelled worms indicated that radioactivity was almost entirely confined to the surface of the worm. Marginal labelling of internal epithelia was observed at orifices and epithelia further inside were not labelled (Fig. 3a & b).

SDS-PAGE analysis of direct immune-precipitates, obtained by adding rabbit anti-AFT serum to extracts of labelled adult worms, regularly showed the presence of four heavily labelled antigens and three weakly labelled antigens (Fig. 4). The antigens were rather labile, showing a tendency on many occasions to give rise to several closely spaced bands, as a result of PMSF resistant partial proteolysis. The major labelled antigens had mol. wts of >150,000, 78,000 +3000, 45,000 ? 3000 and 22,000 f 1000. The values represent the mean f S.D. of nine or more separate determinations. The mol. wt. of the largest antigen could not be estimated accurately due to the absence of suitable mol. wt. markers for this region of the gel. Minor antigens of mol. wts 102,000 & 3000, 59,000 + 2000 and 35,000 * 2000 (mean 2 S.D. of three or more determinations) were frequently observed. Control rabbit anti-ovalbumin-ovalbumin immune-precipitates carried out on equal aliquots of the same labelled extracts possessed, after washing, one-third to one-quarter the amount of radioactivity associated with specific precipitates (generally 300 c.p.s. against 1000 c.P.s.). Analysis of the control precipitates by SDS-PAGE revealed the presence of small amounts of the 45,000 and 22,000 mol. wt. antigens that were also present in the Control precipitates also specific precipitates. material showed considerably less radioactive migrating with the dye front in comparison to specific precipitates. In attempts to vary the conditions of radiolabelling and detergent extraction, adult worms were labelled and washed in Hank’s medium and Nonidet P40 was

I.I.P. VOL. 12. 1982

Surface

antigens

on Schistosoma mansoni

455

(a FIG. 3. Autoradiographs showing the surface localisation of Itz5 1a b e 11e d molecules after lactoperoxidase catalysed iodination of adult worms. (a) Section of the worm at high magnification showing the specific radiolabelling of the tegument. (b) Section at lower magnification at one end of the worm.

-130K -68K -43K

-18AK

A’“r AC AC, AC (HI

(PI

An A”, 41 (HI

A9

(PI

Fro. 4. Surface 1tz5 labelled antigens on adult worms. A, and A, are rabbit anti-AFT precipitates of 1% sodium cholate and I % Nonidet P40 extracts of labelled worms. AC, and An, are the respective control precipitates obtained with rabbit anti-ovalbumin and ovalbumin. H and P denote experiments where the labelhng was carried out in Hank’s medium and PBS respectively.

J.

456

SHAH

and R.

RAMASAMY

I.J.P. VOL. 12. 1982

0

i

i

i

i

TIME (HOURS) FIG. 6. Kinetics of the release of radioactive 1125 labelled adult worms incubated in (-

lb material from ) PBS and

(----------) Hank’s medium. a

b

FIG. 5. The presence of 1125 labelled lactoperoxidase in detergent extracts of labelled adult worms. (a) Labelled protein bound by rabbit anti-ovalbumin serum and (b) labelled protein bound by rabbit anti-lactoperoxidase serum.

used for detergent extraction of labelled proteins. These variations in procedure did not influence the observed pattern of surface antigens (Fig. 4). Centrifugation of the parasite extracts at 16,000 g for 30 min prior to immune-precipitation and the washing of immune-precipitates with ether and ethanol were carried out in attempts to remove nonspecifically binding labelled material. However, these procedures also did not produce a change in the pattern of surface antigens.

dependent on whether the worms were incubated in Hank’s medium or PBS (Fig. 6). Immune-precipitation of the medium after a 24 h incubation revealed the presence of surface antigens in the medium. The electrophoretic pattern of antigens in the medium (Fig. 7) was not identical to the antigens observed in parasite extracts (Fig. 4). This is probably due to a combination of selective release and proteolysis of the antigens. 4

A

Presence of 112s labelled lactoperoxidase in parasite extracts 1125labelled lactoperoxidase was specifically bound by rabbit anti-lactoperoxidase serum from cholate extracts of adult worms labelled by the lactoperoxidase technique. Labelled material was not bound by rabbit anti-ovalbumin serum used as a control. The labelled lactoperoxidase migrated with a mol. wt. of 76,000 in 10% gels (Fig. 5). Similar experiments revealed that 1125 labelled lactoperoxidase was also present in extracts of labelled schistosomula and cercariae. Turnover of surface antigens on adult worms 1125 labelled adult worms released radioactive material with biphasic kinetics into the medium. There was a rapid release of 25% of the initial radioactivity in 2 h followed by a much slower rate of release of the balance. The pattern of release was not

FE. 7. 1’25 labelled surface antigens released by adult worms into the incubation medium. (A) Medium precipitated with rabbit anti-AFT serum and (A,) medium precipitated with rabbit anti-ovalbumin and ovalbumin.

I.J.P. VOL.12. 1982

Surface

A

A1

antigens

S

457

on Schistosoma mansoni

S,

c

c,

Ab Abl

‘b

‘bl

130K -

43K

-

18.4K -

FIG. 8. Common surface antigens on cercariae, schistosomula and adult worms. A, S and C are extracts of adult worms, schistosomula and cercariae labelled by lactoperoxidase catalysed iodination and precipitated with rabbit anti-AFT serum. A,, St and C, are control rabbit anti-ovalbumin-ovalbumin precipitates. At, and S, are rabbit anti-AFT precipitates of extracts of adult worms and schistosomula labelled

with the Bolton-Hunter

reagent. Abl and Sbl are the corresponding rabbit anti-ovalbumin-ovalbumin.

Common surface antigens on larval and adult forms The patterns of labelled surface antigens observed when cholate extracts of labelled cercariae, schistosomula and adult worms were immune-precipitated with rabbit anti-AFT serum is shown in Fig. 8. Control precipitations were performed with ovalbumin and rabbit anti-ovalbumin serum. The results showed that the >150,000, 78,000, 45,000 and the 22,000 mol. wt. antigens present on adult worms were also present on cercariae and schistosomula. Particularly prominent in this respect was the >150,000 mol. wt. antigen. A tendency of this and other surface antigens to partially proteolyse and thus give rise to several closely spaced labelled bands, despite the use of PMSF, was also observed. The quantity of labelled antigens precipitated from extracts of cercariae was always less than that from extracts of schistosomula and adult worms, although approximately equal amounts of radioactivity were initially present in the extracts used for immuneprecipitation. Immune-precipitations carried out with schistosomula and adult worms labelled with the BoltonHunter reagent also showed the presence of common antigens, but the amount of labelled antigens detected, particularly the >150,000 mol. wt. antigen, was less than after lactoperoxidase catalysed labelling (Fig. 8). The presence of common surface antigens on cercariae, schistosomula and adult worms was also investigated using sera from infected patients and control normal human sera. The results from these experiments were complicated by the presence of

control

precipitates

with

several labelled proteins in the control normal human serum-rabbit anti-human IgG immune-precipitates (Figs. 9 & 10). This made the unequivocal identification of many of the labelled proteins present in specific precipitates as antigens difficult. However the >150,000 mol. wt. protein was clearly identified as a specific antigen on all three parasite forms by the use of S7 and S12 sera. Serum S7 also reacted strongly with a diffusely labelled band of material of average mol. wt. 35,000 on cercariae, schistosomula and adult worms. Rabbit anti-AFT and S12 reacted more weakly with this labelled material.

130k66k43k34.7k-

24k-

16.4k-

i Al

A2

1 ~

Sl

Cl

cz

FIG. 9. Autoradiographic pattern of surface antigens on cercariae, schistosomula and adult worms detected with S7 serum. Al, S1 and Cl are specific precipitates of labelled extracts of adult worms, schistosomula and cercariae. A2, S2 and C2 are the corresponding control precipitates obtained using normal human serum.

458

130k68k43k34.7k-

24k-

18.4k-

r

J. SHAH and R. RAMASAMY

injected with AFT, they developed antibodies to the surface of schistosomula that could be demonstrated by indirect immunofluorescence staining of viable schistosomula. In several experiments such mice failed to show significant, reproducible immunity to infection with cercariae. However a significant degree of protection was often observed after injection of Freunds complete adjuvant alone (Table 1). DISCUSSION

i

Al

s2

A2

FIG. 10. Autoradiographic pattern of surface antigens on cercariae, schistosomula and adult worms detected with S12 serum. Al, Sl and Cl are specific precipitates of labelled extracts of adult worms, schistosomula and cercariae. A2, S2 and C2 are the corresponding control precipitates obtained using normal human serum.

The presence of common surface antigens was also investigated by immunofluorescence. Freshly isolated, viable parasites were used to ensure that only surface labelling was observed. The rabbit antiAFT serum reacted with the surfaces of cercariae and schistosomula, in addition to adult worm surfaces, at dilutions of up to 1 : 125. Control rabbit antisera to ovalbumin and normal mouse serum showed some reaction at 1 : 5 dilution but not at 1 : 25 or 1 : 125. Similarly the sera from infected patients, S7 and S12 showed a specific reaction at dilutions of 1 :25 and 1 : 125, in comparison with normal human serum, with the surfaces of cercariae, schistosomula and adult worms. Attempts to induce protection The presence of common schistosomula and adult worms be possible to vaccinate against adult worm surface material.

with worm teguments surface antigens on suggested that it may schistosomiasis using When mice were

TABLE l--E~~t~r

Group A B C D

I.J.P. VOL. 12. 1982

Lactoperoxidase catalysed iodination proved to be a relatively simple and inexpensive procedure for surface labelling schistosomes. While the labelling conditions, particularly the concentration of hydrogen peroxide used, are toxic to parasites such as bloodstream trypanosomes (Alemu & Ramasamy, unpublished observations), schistosomes were not visibly affected by the procedure. Adult worms remained motile and electron microscopy showed that the schistosome tegument was intact after labelling. Autoradiography of sections of 1125 labelled worms showed that radioactivity was largely confined to the surface of the worm. No labelling of the internal epithelial surfaces, as a result of the presence of orifices such as the mouth, the gonopore and the excretory pore, was observed. Some labelling was seen at the orifices themselves. The 1125labelling is therefore largely restricted to molecules present on the outside surface of the worm tegument. It is reasonable to assume that the radiolabelling of cercariae and schistosomula followed a similar pattern. The use of lactoperoxidase technique had a major drawback in that a considerable amount of selflabelled lactoperoxidase remained associated with the parasites after washing. This was extracted, together with the labelled parasite proteins, by 1% solutions of sodium cholate and Nonidet P40. The labelled lactoperoxidase in parasite extracts was bound by rabbit

0~ IMMUNISA~WN WITH AFT IYFb< Tl”N

No. of surviving mice 7 8 7 4

Antibody to schistosomular surface _ + +

ON RESISTANCE TO

No. of worms recovered -t S.D. 100 65 75 77

? 2 + *

43 48 45 41

Nine mice were initially taken for each group. Mice in group A were injected three times with PBS. Group B was injected once with Freund’s complete adjuvant followed by two injections of PBS. Group C was given three injections of 50 worm equivalents of AFT in PBS. Group D was given one injection of 50 worm equivalents of AFT in Freund’s complete adjuvant followed by two injections of AFT in PBS. The differences in worm recovery ol A vs C or D was not significant while that of A vs B was significant al the 1% level, when analysed by the Student’s I test.

I.J.P.voL.

12. 1982

Surface antigens on Schisfosoma mansoni

anti-lactoperoxidase antibodies. Observations made with Leishmania promastigotes during radiolabelling experiments also showed that labelled lactoperoxidase remains associated with the cells, although in much smaller quantities than detected in the present experiments (Ramasamy & Jamnadas, unpublished observations). Therefore, the presence of labelled lactoperoxidase associated with cell surfaces has to be taken into account in interpreting all experiments where lactoperoxidase is used for surface radiolabelling. The mechanism of association of the lactoperoxidase with schistosomes is not clear. This may be due to the physical trapping of protein in the ‘schist’ of adult worms and other surface orifices. It is also possible that iodinated lactoperoxidase binds to membrane proteins as a result of an increased tendency to form non-covalent interactions after iodination. The prominent 76,000 mol. wt. labelled protein detected in SDS extracts of the parasite after lactoperoxidase catalysed iodination is therefore 1125 labelled lactoperoxidase. The full significance of the presence of labelled protein of similar mol. wt. after lactoperoxidase catalysed surface iodination of S. mansoni had not been previously realised (Ramasamy, 1979; Snary, Smith & Clegg, 1980). A comparison of the Bolton-Hunter reagent with the lactoperoxidase technique revealed that the two methods label different but overlapping populations of protein molecules. The reasons for this difference were not investigated. It is probable that the BoltonHunter reagent, which reacts with a and E amino groups labels surface proteins on proteins, differently from lactoperoxidase catalysed iodination where tyrosine residues are labelled. The greater success of the Bolton-Hunter reagent and the corresponding failure of lactoperoxidase for labelling adult worms reported in the experiments of Hayunga et al. (1979a) may reflect minor variations in technique. We have observed that lactoperoxidase catalysed surface iodination is very dependent on factors such as temperature, source of enzyme, time of labelling and other minor variations in the labelling procedure. Immune-precipitation of 1125labelled adult worm extracts with rabbit anti-AFT serum revealed surface antigens of mol. wts >150,000, 102,000, 78,000, 59,000, 45,000, 35,000 and 22,000. Experiments with sera from patients also showed the presence of antibodies to the >150,000 mol. wt. antigen in the sera. The 45,000 and 22,000 mol. wt. antigens were weakly associated with control rabbit anti-ovalbuminovalbumin precipitates. A number of labelled proteins also co-precipitated with control normal human serum-anti-human IgG precipitates. There are several possible explanations for the association of labelled parasite proteins with control immuneprecipitates. This may be a reflection of the tendency of hydrophobic membrane proteins to precipitate from detergent solution. Alternatively these proteins could have F< receptor like activity and therefore

459

react with the IgG in immune-precipitates. F, receptors have been detected on schistosomula (Torpier, Capron & Ouaissi, 1979) and adult worms of S. mansoni (Tarleton & Kemp, 1981). In additional experiments we have been able to demonstrate the presence of the >150,000, 102,000, 78,000, 59,000, 45,000, 35,000 and 22,000 mol. wt. antigens on both male and female S. mansoni worms and worms from different geographically isolated regions of Kenya (Shah, Ramasamy & Kinoti, unpublished observations). Our results on the adult worms compare well with the results of Hayunga et al. (1979b) who were able to identify antigens of 100,000, 78,000, 68,000, 60,000, 43,000, 36,000, 30,000, 26,000 and 20,000 mol. wt. on adult worms. Hayunga et al. (1979b) also reported the detection of a >100,000 mol. wt. glycoprotein which probably corresponds to the >150,000 mol. wt. antigen detected in our experiments. Carbohydrate labelling procedures carried out in our work also suggest that the >15O,ooO mol. wt. antigen is a glycoprotein. The detection of surface antigens of the same mol. wt. on cercariae, schistosomula and adult worms, by a rabbit antiserum to adult worm antigens shows that the labelled antigens are common to all three forms of S. mansoni. The relevance to human schistosomiasis is shown by the presence of antibodies to the common surface antigens in the sera of patients carrying active infections. The presence of common surface antigens on the three forms of the parasite was confirmed by the surface immunofluorescence studies. It is also supported by the detection of common antigens in AFT and detergent extracts of cercariae and schistosomula by Ouchterlony immunodiffusion using sera from patients and the rabbit anti-AFT serum (Shah & Ramasamy, 1981). The presence of common surface antigens on larval and adult parasites provides part of the explanation for the phenomenon of concomitant immunity in schistosomiasis (Smithers & Terry, 1969). Our results and those of other workers (Kusel et al., 1975) show that surface antigens are released from the adult worms into the medium in vitro. The antigens released from the worms into the bloodstream in vivo can stimulate the host to produce antibodies. The antibodies can then bind to the surfaces of the invading schistosomula of a secondary infection and bring about their destruction (Clegg & Smithers, 1972; Butterworth et al., 1975, Capron et al., 1975; Perez & Smithers, 1977). The presence of antibodies to adult worm surfaces in infected patients also raises the question of how the worms escape destruction by antibody dependent immunological mechanisms. Our experiments with rabbit antiserum to normal mouse serum produced no evidence for the presence of mouse serum antigens on the parasite surface. Hayunga et al. (1979b) were also unsuccesful in showing host antigens on adult worm surfaces. It is possible that host antigens that are acquired by schistosomes (Smithers et al., 1969; Coelho, Gazzi-

J. SHAH and R. RAMASAMY

460

nelli & Pellegrino, 1980) are either washed away from the worms during our experiments or are not detected as antigens by our rabbit antiserum to mouse serum. Since AFT contained antigens that were also present on schistosomular surfaces, it was felt worthwhile to attempt the immunisation of mice against schistosomiasis with AFT. Mice injected with AFT developed antibodies to the surfaces of schistosomula that were detectable by immunofluorescence. However such mice were not protected against infection. There are several explanations for the failure to induce protective immunity. It is possible, for example, that cells such as eosinophils may not be induced in the immunised mice in sufficient numbers to aid the killing of schistosomula. A further possibility is that IgG sub-classes not involved in complement and antibody dependent, cell mediated killing of schistosomula (Ramalho-Pinto, De Rossi & Smithers, 1979) are produced during immunisation and these then function as ‘blocking’ antibodies preventing the destruction of schistosomula. Therefore immunity to infection may depend on a fine balance of critical cells and antibodies. During the operation of concomitant immunity, the presence of living adult worms in the host may produce the right mixture of cells and antibodies to bring about the destruction of the schistosomula of a secondary infection. The failure of adult worm extracts to induce protective immunity, a phenomenon that has been widely observed (Phillips & Reid, 1980), therefore needs further investigation. Acknowledgemenfs-This

work was supported by grants

from the International Atomic Energy Agency (2592-RB) and the Dean’s Committee of the University of Nairobi. We are grateful to the Wellcome Trust Laboratories, Nairobi for the provision of parasite material.

REFERENCES BUTTERWORTH A. E., STURROCK R. F., HOUBA V., MAHMOUD, A. A. F., SHER A. & REES P. H. 1975. Eosinophils as mediators of antibody dependent damage to schistosomula in vitro. Nature (London) 256: 727-729. BONNER W. M. & LASKEY R. A. 1974. Fluorographic detection of 3H, 14C and YS in acrylamide gels. European Journal of Biochemistry 46: 83-88. CAPRON A., BAZIN H., DESAINT J. P. & CAPRON M. 1975. Specific IgE antibodies in immune adherence of normal macrophages to Schisfosoma mansoni schistosomules. Nature (London) 253: 474-475. CLEW J. A. & SMITHERS S. R. 1972. The effect of immune rhesus monkey serum on schistosomula of Schisfosoma mansoni during cultivation in vitro. International Journal for Parasitology 2: 79-98. COELHO P. M. Z., GAZZINELLI G. & PELLEGRINO J. 1980. Schistosoma mansoni: host antigen occurrence on worms recovered from laboratory vertebrate animals. Parasitology 81: 349-354. GAHMBERG C. G. & HAKAMORI S. 1973. External labelling of cell surface galactose and galactosamine in glycolipid

I.J.P. VOL. 12. 1982

and glycoprotein of human erythrocytes. Journal of Biological Chemistry 248: 43 1 l-43 17. HAYUNGA E. G., MURRELL K. D., TAYLOR D. W. & VANNIER W. E. 1979a. Isolation and characterisation of surface antigens from Schistosoma mansoni. 2. Antigenition of techniques for radioisotope labelling of surface proteins from adult worms. Journal of Parasitology 65: 488-496. HAYUNGA E. G., MURRELL K. D., TAYLOR D. W. & VANNIER W. E. 1979b. Isolation and characterisation of surface antigens from Schistosoma mansoni. 2. Antigencity of radiolabelled proteins from adult worms. Journal of Parasitology 65: 497-506. HOCKLEY D. J. & MCLAREN D. J. 1973. Schistosoma mansoni: Changes in the outer membrane of the tegument during development from cercaria to adult worm. International Journal for Parasitology 3: 13-25. KESSLER S. W. 1975. Rapid isolation of antigens from cells with staphylococcal protein A antibody absorbent: Parameters of the interaction of antibody-antigen complex with protein A. Journal of Immunology 115: 16171624. KUSEL J. R. 1972. Protein composition and protein synthesis in the surface membranes of Schisfosoma mansoni. Parasitology 65: 55-69. KUSEL J. R., MACKENZIE P. E. & MCLAREN D. J. 1975. The release of membrane antigens into culture by adult Schistosoma mansoni. Parasitology 11: 247-259. LAEMMLIU. K. 1971. Changes of structural proteins during the assembly of the head of the bacteriophage T4. Nature (London) 227: 680-685. MARCHALONIS J. J., CONE R. E. & SANTER V. 1971. Enzymatic iodination. A probe for accessible surface proteins on normal and neoplastic lymphocytes. Biochemical Journal 124: 921-927. OAKS J. A. & LLJMSDENR. D. 1971. Cystological studies on the absorbtive surfaces of cestodes. 5. Incorporation of carbohydrate-containing macromolecules into tegument membranes. Journal of Parasitology 57: 1256-1268. PEREZ H. & TERRY R. J. 1973. The killing of adult Schistosoma mansoni in vifro in the presence of antisera to host antigenic determinants and peritoneal cells. International Journal for Parasitology 3: 499-503. PEREZ H. & SMITHERS S. R. 1977. Schistosoma mU/,soni in the rat: the adherence of macrophages to schistosomula in vitro after sensitisation with immune serum. International Journal for Parasitology 7: 3 15-320. PHILLIPS D. R. & MORRISONM. 1970. The arrangement of proteins in the human erythrocyte membrane. Biochemical and Biophysical Research Communications 40: 284-289. PHILLIPS S. M. & REID W. A. 1980. Schistosoma mansoni: Immune response to normal and irradiated cercariae or soluble stage-specific surface immunogens. International Journal of Nuclear Medicine and Biology 7: 173- 186. RAMALHO-PINTO F. J., DE Rossr R. & SMITHERS S. R. 1979. Murine Schistosomiasis mansoni: anti-schistosomula antibodies and the IgG sub-classes involved in the complement and eosinophil-mediated killing of schistosomula in vitro. Parasite Immunology 1: 295-308. RAMASAMYR. 1979. Surface proteins on schistosomula and cercariae of Schistosoma mansoni. International Journal for Parasitology 9: 49 l-493. RAMASAMY R., JAMNADAS H. & MUTINGA M. J. 1981. Proteins and surface proteins of Leishmania promastigates and their possible relevance to the characterisation

I.I.P. VOL. 12. 1982

Surface

antigens

on Schistosoma

of strains. International Journal for Parasitology 11: 387-390. RUPPEL A. & CIOLI D. 1977. A comparative analysis of various development stages of Schistosoma mansoni with respect to their protein composition. Parasitology 75:

339-343. SHAH J. & RAMASAMY R. 198 1. Schistosoma mansoni: Common antigens on adult worm teguments and cercariae detected by immunodiffusion. East African Medical

Journal 58: 330-333. SMITHERS S. R. & TERRY R. J. 1969. The immunology of schistosomiasis. Advances in Parasitology 7: 41-93. SMITHERS S. R., TERRY R. J. & HOCKLEY D. J. 1969. Host

antigens

mansoni in schistosomiasis.

461

Proceedings of the Royal

Society B 171: 483-494. SNARY D., SMIIITH M. A. & CLECX J. A. 1980. Surface proteins of Schistosoma mansoni and their expression during morphogenesis. European Journal of Immunology 10: 573-575. TARLETON R. L. & KEMP W. M. 1981. Demonstration of IgG-Fc and C3 receptors on adult Schistosoma mansoni.

Journal of Immunology 126: 379-384. TORPIER G., CAPRON A. & QLJAISSI M. A. 1979. Receptor for IgG (Fc) and human beta 2-microglobulin on S. mansoni schistosomula. Nature (London) 278: 447-449.