EXPERIMENTALPARASITOLOGY

Schistosoma

70,293-304 (19%)

japonicum:

Monoclonal Antibodies to the M, 26,000 Schistosome Glutathione S-Transferase (Sj26) in an Assay for Circulating Antigen in Infected Individuals K.M.

DAVERN, W.U.Tw,* N. SAMARAS, D.P. GEARING, B. E. HALL, E.G. GARCIA,* AND G. F. MITCHELL

The Walter and Eliza Hall Institute of Medical Research, Melbourne, *Department of Parasitology, College of Public Health, University 1000 Manila, Philippines DAVERN, K. M., TIU, W. U., SAMARAS, N., GEARING, E. G., AND MITCHELL, G. F. 1990. Schistosoma japonicum:

Victoria 3050, Australia, and of the Philippines Manila,

D. P., HALL,

B. E., GARCIA,

Monoclonal antibodies to the M, 26,000 schistosome glutathione S-transferase (Sj26) in an assay for circulating antigen in infected individuals. Experimental Parasitology 70, 293-304. Two monoclonal antibodies have been produced that bind to separate epitopes on the M, 26,000 glutathione S-transferase (GST) of Schistosoma japonicum worms (Sj26). Both antibodies have been used in an enzyme immunoassay (EIA) with sera from infected individuals from the Philippines. Relatively high signals were obtained with set-a from some, but not all, individuals who are positive for fecal eggs. Evidence was obtained that the material detected by the monoclonal antibodies was present in minute amounts and in some sera was bound in a complex with phosphorylcholine-containing molecules. It could not be absorbed by reaction with glutathione-agarose columns. There was no detectable immunoglobulin in the complex. The possibility exists that the complexes are composed of schistosome GST, or fragments, and damaged tegumental lipids shed as a result of surface immune attack. However, the presence of the native Sj26 molecule has not been proven. More detailed longitudinal studies in endemic areas are required to determine whether the assay can be used as an indicator of acquired resistance (“concomitant immunity”) and whether it will be useful in the search for immunological correlates of this resistance in humans. 8 1990 Academic Press, Inc.

INDEX DESCRIPTORS AND ABBREVIATIONS: Schistosoma japonicum; Trematode; Adult worm extract; (AWE); Bovine serum albumin (BSA); Competitive radioimmunoassay (CRIA); Murine epidermal growth factor (EGF); Enzyme immunoassay (EIA); Enzymelinked immunosorbent assay (ELISA); Freunds complete adjuvant (FCA); Glutathione Stransferases purified from Fasciola hepatica adult worms (FhGST); Recombinant molecule comprising the first 69 amino acids at the 5’ end of the Sj26 molecule (Fragment A); Reduced glutathione (GSH); Horseradish peroxidase (HRPO); Immunoradiometric assay (IRMA); Murine leukemia inhibitory factor (LIF); Monoclonal antibody (MoAb); Phosphate-buffered saline, pH 7.3 (PBS); Phosphorylcholine (PC); S. japonicum GST (Sj26) produced from the pGEX vector (pGEX-1); EGF expressed in the pGEX vector (EGF-GEX); LIF expressed in the pGEX vector (LIF-GEX); LIF molecule expressed with, at its C-terminus, 8 amino acids from the C-terminus of Sj26 (CT-LIF); Praziquantel (PZQ); Radioimmunoassay (RIA); Recombinant near native Sj26 (rSj26); Sj26-B-Galactosidase fusion protein (Sj26FP); Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); Sj26, Sj28, Sm26, and Sm28, M, 26,000 and M, 28,000 GST isoenzymes of S. japonicum and S. mansoni.

INTR~DIJCTI~N

vaccine against schistosomiasis (James and Sher 1986; Capron et al. 1987; Bergquist 1987; Simpson and Cioli 1987; Smithers 1987). Glutathione S-transferase isoenzymes (GSTs) of schistosomes are of some

Several molecules of schistosomes have been identified that may be useful ingredients in a multicomponent, defined-antigen 293

0014-4894/90 $3.00 Copyrisht 8 l!NOby Academic Press,Inc. All rights of reproduction in any form reserved.

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interest in this regard (Mitchell 1989) and at 1986). Subsequent SDS-PAGE analysis of these purileast two are available as enzymatically ac- hed GSTs showed them to be resolved into two bands of 44, 26,000 and M, 28,000 (Sj26 and Sj28 for S. tive, native or near-native molecules pro- japonicum, and Sm26 and Sm28 for S. mansoni) (Tiu duced in Escherichia coli-namely, Sj26, a et al. 1988). GST purified in the same way from FasM, 26,000 GST of Schistosoma japonicum ciola hepatica adult worms was kindly provided by Professor Cohn Chapman, Victorian College of Phar(Philippines) (Smith et al. 1988), and Sm28, a M, 28,000 GST of S. mansoni (Taylor et macy, Melbourne. Human GSTs were provided by Dr. Philip Board, John Curtin School of Medical Real. 1988). Both S. japonicum and S. man- search, Australia. Human GSTs used were GSTl pusoni contain two GSTs of M, 26,000 and M, ritied from liver. recombinant GST2 from liver. and 28,000 as analyzed by SDS-PAGE (Tiu et GST3 purified from lung. Recombinant Sj26 antigens al. 1988).

In schistosomiasis japonica, the GSTs have been found to be poor immunogens during human infection. Low titers of apparently low-affinity anti-Sj26 antibodies can be detected using a sensitive ELISA in sera of individuals both with and without schistosome infection (Lightowlers and Mitchell 1989). However, high-titered antiSj26 antibodies can be induced in animals by immunization, in the presence of strong adjuvants, with either recombinant Sj26 or adult worm GST (Davern et al. 1987; Tiu et al. 1988). Because of the low apparent immunogenicity of Sj26 in humans, we have addressed the question of whether Sj26 can be detected as a circulating antigen in the sera of Philippine individuals infected with S. japonicum. For this purpose, a double monoclonal antibody sandwich enzyme immunoassay (EIA) was developed. Preliminary results suggest that a positive signal in this assay with sera from individuals living in endemic areas may be an indicator of acquired resistance. MATERIALSANDMETHODS Parasite life cycles were maintained as described previously (Wright et al. 1988). Rabbits used for production of antisera or as a source of S. japonicum (Philippines) were from outbred stocks maintained conventionally. Mice were derived from a specific pathogen-free facility but maintained conventionally from approximately 6 weeks of age. Glutathione S-transferases (GSTs) were purified from S. japonicum or S. mansoni crude adult worm extract (AWE) by affinity chromatography on glutathione (GSH)-agarose columns (Sigma Chemical Co., St. Louis, MO) as previously reported (Smith et al.

produced in E. coli and used in this paper are the Sj26-B-galactosidase fusion protein (Sj26FP) (Smith et al. 1986), the near native recombinant Sj26 (rSj26) (Smith et al. 1988), the GST protein from the pGEX vector (Medos, Australia) which contains an additional 10 amino acids at the C-terminus of Sj26 (pGEX1) (Smith and Johnson 1988) as well as various composite antigens produced as fusion proteins in pGEX. These include a portion of a 65-kDa protein of Mycobacterium bovis (a gift of Drs. A. Radford and P. Wood, CSIRO Division of Animal Health, Melbourne), a Plasmodium falciparum protein, Ag513 (Smythe et al. 1988) murine epidermal growth factor (EGF-GEX) (D. P. Gearing and A. W. Burgess), and murine leukaemia inhibitory factor (LIF) (Gearing et a/. 1987). A fragment encoding the first 69 amino acids at the N-terminus of Sj26 was also expressed and used in dot blot analyses, and a recombinant clone encoding 8 amino acids (GGGDHPPK) from the C-terminus of rSj26 added to the C-terminus of a clone of murine leukaemia inhibitory factor (CT-LIF) was also used. The C-terminal sequence of Sm26, GGGDAPPK, was provided by Drs. R. Harrison and G. Newport, UCSF, U.S.A. Rabbits were immunized with SjAWE, SjGST puritied from adult worms, or rSj26 in Freund’s complete adjuvant (FCA) followed by at least two aqueous boosts. Female mice of WEHI 129/J and BALB/K strains were immunized with rSj26, a portion of the M. bovis 65-kDa protein expressed in pGEX or P. fulciparum Ag513 in pGEX. After an aqueous boost, sera were collected and assayed for antibodies to Sj26 in a protein A-ELISA. Human sera were collected from patients aged between 7 and 33 years as part of studies in the Sorsogon and Leyte regions of the Philippines. At the time of sera collection, all were shown to be infected by detection of fecal eggs. All were bled and, on the same day, treated with praziquantel (PZQ). Individuals were provisionally classified as resistant or susceptible to reinfection by measuring fecal egg counts. In most cases, multiple samples were collected and several different fecal egg counts were performed in order to classify individuals as resistant or susceptible. In the case of Sorsogon individuals, follow-up examinations

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were performed at 4 to 8 months and then 10 to 20 months after serum collection and PZQ treatment. Leyte individuals were assessed at yearly intervals for between 2 and 3 years after serum collection and treatment. An estimate of exposure was recorded in the case of Sorsogon individuals based on the average number of reported water contacts in the field per week, and sera from individuals with reported exposure of more than once per week were used in these studies. The human sera from Leyte were provided by Dr. Remigio Olveda of the Research Institute for Tropical Medicine, Alabang, Philippines. The positive human serum pool used in some experiments was composed of five sera which gave a signal, at a dilution of 15, greater than 1.2 OD units above control sera in the EIA. Negative control sera were collected from healthy Melbourne individuals and used as a pool. In some assays, control sera were from Philippine individuals not residing in schistosomiasis endemic areas or from Papua New Guinea patients living in an area endemic for filariasis. Monoclonal antibodies MoAbSO-1 and MoAb250-7 were produced by fusion of the myeloma line SP2/0 and spleen cells from BALBlc mice immunized with Sj26FP in FCA, followed by three aqueous boosts, the last one 4 days prior to fusion. BALB/c mice, although low responders to Sj26, produce significant levels of antibody when immunized with high amounts of antigen (Davem et al. 1987). Supematants were assayed by ELISA on both SjAWE and SjGST using horseradish peroxidase (HRPO)-conjugated sheep anti-mouse immunoglobulin (Silenus Laboratories, Melbourne, Australia) as readout. As both monoclonal antibodies are of the IgM isotype, they were purified from ascites by ammonium sulfate precipitation as described previously (Cruise ef al. 1981) and, for MoAb250-7 which binds to protein A, subsequent purification on protein A-Sepharose (Pharmacia, Sweden) in some experiments. Purified MoAbs were conjugated with HRPO (Sigma) (Goding 1986) or were iodinated, as was the protein A (Sigma) which was used in Western blotting analyses, using the chloramine T method (Greenwood et al. 1963). Western blotting analyses were performed on SjAWE or SjGST after SDS-PAGE, transfer to nitrocellulose sheets, and incubation of nitrocellulose strips with lZ51-MoAbs or 1:500 dilutions of rabbit antisera, followed by “‘I-protein A. All incubations were performed in 5% skim milk in PBS (BLOTTO). Strips were dried and autoradiographed against Curix RP2 film (Agfa, Belgium). Dot blot analyses were performed by adding equal amounts of proteins, usually in PBS, to nitrocellulose paper, allowing them to dry, blocking with BLOTTO, and then adding “‘1-MoAbs diluted in BLOTTO. After incubation, washing with BLOTTO, and drying, the dot blots were set up for autoradiography.

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Direct binding radioimmunoassays and ELISA assays were performed in %-well polyvinylchloride microtiter pIates (Dynatech Laboratories, Virginia, U.S.A.) (Davem et al. 1987). Antigen, at 5-10 pg/ml in PBS, was coated at 50 @well for 3 hr or overnight. After blocking the wells with 0.5% bovine serum albumin (BSA) solution, wells were incubated with labeled MoAb or dilutions of sera followed by labeled protein A or the appropriate anti-immunoglobulin. For ELISA assays, the substrate 2,2’-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (Sigma) was added and signals were measured as described in Davem et al. (1987). Competitive RIAs (CRIAs) were performed with a limiting concentration of coating antigen, followed by overnight incubation of dilutions of sera or unlabeled MoAb together with “‘1-MoAb. Enzyme immunoassays (EIA) for detection of circulating antigen were performed in microtiter plates coated for 5 hr with 5-10 wLg/ml of purified MoAb, usually MoAbSO-1. After blocking with BSA, human sera or rSj26 were diluted in 0.5% B&A/0.05% Tween 20/PBS and added to the wells and allowed to incubate overnight. The next morning the plates were washed, HRPO-conjugated MoAb250-7 was added to the wells, and incubation was continued for 5-7 hr after which time the plates were washed and substrate was added. All incubations were performed at 37°C. In some assays, the EIA was performed with HRPO-protein A or HRPO-sheep anti-human Ig (Silenus) replacing HRPO-MoAb250-7 as the readout. In some experiments, several different MoAbs were used to coat the plates, all at 5-10 &ml, followed by human sera and then HRPG-MoAb250-7. Control MoAbs used were Gibl3, 32D, and 2F (all IgM anti-phosphorylcholine (PC)), HOPCS (IgA anti-PC), CB22, G9, and aPCH5G (all IgG anti-PC), FM1.35 (IgM anti-human granulocyte membrane), WIClO8 (IgM anti-leishmania major glycoconjugate), 3H15H (IgM anti-TEPC15 idiotype), and 7-l-3 (IgG anti-azobenzene-arsonate). Purified MoAbs were gifts from Drs. Grant Morahan, Karen Forsyth, and Emanuela Handman of this institute and from Phillip O’Connell, Royal Melbourne Hospital. Attempted depletion of the circulating antigen from human sera was performed using GSH-agarose, protein A-Sepharose, or affinity columns of CNBractivated Sepharose 4B (Pharmacia) coupled, according to manufacturer’s instruction, with MoAbSO-1, MoAb250-7, or sheep anti-human Ig. To 100 ~1 of packed beads, 1 ml of human serum or 100 pg of rSj26 was added and incubated for 2 hr on ice, followed by 2 hr at room temperature with occasional mixing. The supematants were collected after incubation and assayed by EIA to measure depletion. The beads were washed three times with 10 ml PBS and used in Westem blotting analyses or were radioiodinated using 100 &i of the Bolton and Hunter reagent (Amersham, U.K.). After extensive washing to remove unbound

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radioisotope, a sample of beads was incubated with 0.1 M glycine/HCl buffer, pH 2.6, to elute material bound to the MoAb. The eluate was analyzed by SDS-PAGE and autoradiography before or after immunoprecipitation using rabbit anti-Sj26 antisera or MoAbs conjugated to Sepharose beads. RESULTS

Specificity of anti-Sj26 monoclonal antibodies. The two monoclonal antibodies,

MoAbSO-I and MoAb250-7, were labeled with ‘251and used to probe Western blots of either S. japonicum adult worm extract (AWE) or GST purified from adult worms by affinity chromatography on glutathioneagarose. It can be seen in Fig. 1(lanes D, E, J, and K) that both labeled antibodies bind to Sj26 with no apparent reactivity with Sj28 or other molecules in the adult worm extract. A control IgM MoAb failed to react with any schistosome antigens (lanes F and Sj AWE

Sj GST

?kl 30

ABCDEF GHIJKL FIG. 1. Autoradiography of Western blots of 5. japonicum AWE (lanes A-F) and S. japonicum GST (lanes G-L) probed with rabbit antiserum to SjAWE (lanes A and G), rabbit anti-SjGST (lanes B and H), rabbit anti-rSj26 (lanes C and I), MoAbSO-1 (lanes D and J), MoAb250-7 (lanes E and K), or control IgM MoAb (lanes F and L). Rabbit antisera incubations were followed by “‘I-protein A and MoAbs were directly iodinated. Molecular weight markers, 20 and 30 kDa, are indicated.

ET AL.

L). Positive control rabbit antisera raised to SjGST recognized both Sj26 and Sj28 (lanes B and H), rabbit anti-rSj26 antibodies recognized Sj26 but not Sj28 (lanes C and I), and anti-SjAWE antibodies detected numerous molecules in AWE, as well as Sj26 and Sj28 (lanes A and G). In Table I, results from various assays determining the cross-reactivity of both monoclonal antibodies are shown in summary form. MoAbSO-1 and MoAb250-7 labeled with 125I were used in radioimmunoassays with purified GSTs from S. japonicum, S. mansoni, and F. hepatica adults. MoAbSO-I bound significantly to FhGST, whereas there was no detectable binding of MoAb250-7. On the other hand, MoAb250-7 showed some binding, though only at high antigen concentrations, to SmGST. In competitive RIAs, the two MoAbs showed weak, but detectable, cross inhibition. These data suggest that the two MoAbs are directed to different epitopes on the Sj26 molecule. This was confirmed using composite Sj26 antigens produced in E. coli. In ELISAs, both MoAbs bound to the near native rSj26 and also to the B-galactosidase-Sj26fusion protein (Sj26FP) which was the antigen used to immunize the TABLE I Binding of MoAbSO-1 and MoAb250-7 to S. japonicum, S. mansoni, and F. hepatica Antigens Antigen

MoAbSO-l0

SjGST SmGST FhGST Sj26FP rSj26 pGEX-1 Fragment A EGF EGF-GEX LIF LIF-GEX CT-LIF

+ + + + + + + -

MoAb250-7” + + + + +

a Results of binding of MoAbSO-1 or MoAb250-7 to various antigen preparations in ELISAs, RIAs, or, in the case of fragment A, in a dot blot assay.

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spleen donors prior to fusion. However, whereas the MoAbSO-1 bound to the pGEX-1 product, which contains an additional 10 amino acids at the C-terminus of Sj26, as well as to various composite antigens produced as fusion proteins in pGEX, MoAb250-7 failed to recognize any of these. This result was confirmed in a dot blot assay where ‘*‘I-MoAb50- 1 bound to both rSj26 and pGEX-1, while 1251MoAb250-7 only reacted with rSj26. In the same assay, neither MoAb bound to a fragment (Fragment A) representing the first 69 amino acids from the H-terminus of Sj26. In a direct binding ELISA, using as the coating antigen recombinant LIF with, at its Cterminus, 8 amino acids (GGGDHPPK) from the C-terminus of Sj26 (CT-LIF), MoAb250-7 gave a strong positive signal, whereas MoAbSO-1 did not bind. These data clearly indicate that the target epitopes of both MoAbs are distinct and that MoAb250-7 binds to an epitope that is dependent on a free C-terminus of the Sj26 molecule and is defined by the 8 C-terminal residues. The loss of an epitope from the pGEX-1 molecule, as well as Sj26 fusion proteins produced in pGEX, as indicated by the failure of MoAb250-7 to bind, may have wider implications in the search for a vaccine candidate that is more immunogenic than rSj26. Sera from mice of WEHI129/J and BALB/K strains immunized with Sj26, as a recombinant protein (rSj26), or as a fusion protein with either M. bovis or P. fulciparum antigens produced in the pGEX-1 vector, were assayed in a protein A-ELISA for antiSj26 antibodies (results not shown). In both mouse strains, the immunogenicity of rSj26 was greater than either of the two Sj26 fusion proteins. For WEHI129/J mice the antibody response in rSj26-immunized mice was at least five times greater, and for BALB/K the response was increased by at least 20-fold over those induced by the fusion proteins. Several other Sj26 fusion proteins have also been tested with disap-

297

pointing results in terms of anti-Sj26 antibody production. Monoclonal antibodies EIA. In an attempt to ascertain if Sj26 can be detected in the serum of infected individuals, a two-site sandwich EIA was devised which employed both monoclonal antibodies in combination. Because the two MoAbs are directed to different epitopes, their combined use in an EIA should decrease the possibility of detecting non-Sj26 antigen, including cross-reactive molecules or anti-idiotype antibodies, which may be detected by using a single MoAb. In competitive RIAs, using sera from infected patients in the Philippines, no significant inhibition of binding to Sj26 of either of the two MoAbs was detected. Thus the two epitopes are of low immunogenicity during infection and should not be masked by any anti-Sj26 antibodies in sera from infected humans. In fact, in direct binding ELISAs using GST purified from adult worms or rSj26 as antigen and HRPO-protein A as readout, antibody titers in sera from infected Philippine patients were very low (results not shown). Sera used in the EIA were collected from 20 selected patients aged between 7 and 33 years residing in the endemic areas of the provinces of Sorsogon and Leyte, Philippines. Sera were collected at a time when all were shown to be infected (i.e., fecal egg count positive). Patients were then treated with PZQ and on subsequent follow-ups were able to be categorized by fecal egg examinations as susceptible (i.e., those who became reinfected) or resistant (i.e., those with no evidence of reinfection). We are aware of the shortcomings of this classification based on an entirely unsatisfactory assessment of infection-namely, eggs in a small sample of feces. However, in most instances for classification, multiple samples were used and several fecal egg counts were performed (see Materials and Methods). The assay was performed in microtiter plates with MoAbSO-1 in solid phase, dilu-

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tions of sera from I:5 added to the wells and enzyme-tagged MoAb250-7 as the readout. The data presented in Fig. 2 indicate there is detectable activity in infected sera when compared with a pool of uninfected Melbourne sera. For several individuals, at high serum concentration, the absorbance in the EIA was high, although the signal dropped off rapidly, so that in most cases by between 1:lO and 1:20 dilution of serum, there was virtually no detectable activity left. Presumably this is an indication of the extremely low levels of the material being detected. Sera from 20 Philippine individuals not residing in schistosomiasis areas were unequivocally negative (W. U. Tiu

1:s

130

Dilutions of sero (Philippines 1 FIG. 2. OD,, results of EIA with MoAbSO-l-coated plates followed by sera, at 13 and 1: 10 dilutions, from 20 Philippine schistosomiasis japonica patients (0) and using HRPO-MoAb250-7 as readout. Results in the EIA with rSj26 (A) and control Melbourne human serum pool (A) are also indicated.

ET

AL.

and E. Cl. Garcia, unpublished observations) as were sera from five Papua New Guinea patients living in a Wuchereria bancrofti endemic area. Three of the five were shown to be positive in the Gib13 IRMA for W. bancrofti infection (Dissanayake et al. 1984). A range of signals is apparent in Fig. 2. Although the numbers of sera are low in each of the categories of putatively susceptible (9 individuals) and putatively resistant to reinfection after PZQ (11 individuals), there is a significantly higher level of detectable activity in the sera from resistant individuals (P < 0.05). Thus 9 of 11 resistants had a reading >1.2 OD units in the EIA compared with 1 of 9 susceptibles. The important point of the data to date is that only some sera from infected individuals are positive in the EIA; more analyses are required to establish whether this is an interesting subset of individuals. The assay worked equally well with MoAbSO-1 or MoAb250-7 in solid-phase and enzyme-conjugated MoAb250-7 as the readout. Using HRPO-MoAbSO-1 as the readout gave lower signals in either assay. MoAbSO-1 was chosen as the coating antibody and HRPO-MoAb250-7 as the readout in assays reported here. It is interesting to note that, for detection of activity in human sera, we have been unable to convert the assay to IRMA, using 1251-MoAbs as the readout. The rSj26 was positive in both assays. Three different isoenzymes of human GSTs, two from human liver and one from human lung, were assayed alongside rSj26 in the EIA and were found to be negative (results not shown). In order to reduce the possibility that anti-idiotype was being detected in the EIA, two monoclonal antibodies of different specificities were intentionally employed. Results shown in Table II provide evidence that anti-idiotype antibodies do not contribute to positive signals in this assay. The EIA was performed using either HRPO-conjugated protein A or HRPOanti-human immunoglobulin (aHIg) replacing MoAb250-7 as the readout. A pool of

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TABLE II Absorbance Units in EIA” MoAb250-I

Anti-human Iis

Protein A

High pool sera l/5 l/10 l/20

1.860 0.885 0.317

0.005 0.005 0.005

0.047 0.002 0.005

rSj26 l/50 l/loo l/200

2.117 1.922 1.548

0.004 0.004 0.004

0.004 0.002 0.002

a OD,, results of EIA on MoAbSO-l-coated plates, followed by incubation with pooled “high positive” S. japonicum infection sera, or rSj26, at various dilutions and using HRPO-MoAb250-7, HRPO-antihuman immunoglobuhn, or HRPO-protein A as readout.

positive human sera and rSj26 were each positive in the double monoclonal antibody EIA. However, in both cases, there was no activity detected using aHIg or protein A. In all three assays, a pool of control Melbourne human sera was negative. The activity detected in the EIA in a pool of positive human sera could be removed by incubating the serum pool with Sepharose beads coupled with either MoAbSO-1 or MoAb250-7 (Fig. 3). Both MoAb-Sepharose beads also successfully depleted the EIA activity from rSj26, as did GSH-agarose beads. However, GSHagarose beads failed to remove a significant amount of the EIA-positive material from the human serum pool. Incubation with control Sepharose beads did not cause any depletion of activity. With the possibility that any Sj26 in serum may already be complexed with GSH, sera were dialyzed extensively against PBS or against 0.05 M Tris, pH 9.5, followed by PBS prior to incubation with GSH-agarose. There was a slight reduction of the signal in the EIA following dialysis against PBS. After incubation with the Tris buffer, pH 9.5, the EIA results for both the positive human serum pool and the rSj26 were reduced. Presumably extensive dialysis at high pH leads to

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degradation of the material. Following dialysis, incubation of sera with GSH-agarose beads failed to remove the remaining material. Results in Fig. 3 also show that incubation with protein A-Sepharose or rabbit anti-human Ig-Sepharose beads did not deplete the signal. This confirms the results in Table II indicating the lack of immunoglobulin in the material being detected in the EIA. Following the successful depletion from serum of EIA activity on MoAb-Sepharose beads, several attempts were made to identify the nature of the material. Incubation of Western blots of positive human sera, or MoAb-Sepharose beads after absorption of the material from positive human sera and probed with a variety of anti-SjGST antibodies, failed to demonstrate the presence of Sj26. After incubation of the beads with the serum pool and extensive washing to remove unbound material, the beads were iodinated and analyzed by SDS-PAGE, either directly or after elution and immunoprecipitation of the bound material. Again Sj26 could not be detected. Interestingly, while checking the specificity of the MoAbs in the EIA, it was found that anti-phosphorylcholine (PC) MoAbs could result in a positive signal in the assay. Positive signals were obtained using the Anti-PC MoAb Gib13 as the coating antibody, followed by dilutions of positive human sera and using HRPO-conjugated MoAb250-7 as the readout (Fig. 4). This result was confined to the positive human serum pool as control sera and rSj26 gave a very low or negative signal. Several anti-PC MoAbs led to detection of positive signals, and all were of the IgM and IgA isotypes. However, results using an IgA anti-PC MoAb have not been included as we have not tested control IgA antibodies in the assay. Control IgM and IgG MoAbs of other specificities were negative under the conditions of the assay. Several IgG anti-PC MoAbs were also negative with all sera tested. When tested individually, most of the human sera that were positive in the 50-

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ET AL.

80

1

2

3

4

5

6

TREATMENT FIG. 3. Calculated percentage depletion of EIA activity after incubation of rSj26 (solid) or positive human serum pool (hatched) following incubation with various absorbents. The preparations used for depletion were MoAbSO-1Sepharose (l), MoAb250-7-Sepharose (2), control BSASepharose (3) GSH-agarose (4), sheep anti-human Ig-Sepharose (5), and protein A-Sepharose (6).

l/250-7 EIA were positive, and at similar levels, in the anti-PC/250-7 EIA. However, this was not invariably the case. Several positive sera gave a low or undetectable signal in the anti-PC/250-7 assay. To date, there has been no serum that was negative in the 50-l/250-7 assay that gave a positive signal in the anti-PC/250-7 assay. DISCUSSION

In this paper, evidence has been presented that two monoclonal antibodies which react with Sj26, a glutathione Stransferase (GST) isoenzyme of S. juponicum, can be employed in a double mono-

clonal antibody sandwich EIA to detect activity in the sera of a proportion of individuals currently infected with S. juponicum. Although the number of sera examined to date is limited, there does appear to be a higher signal in sera from individuals who, after PZQ treatment, are resistant to reinfection as assessed by fecal egg counts in longitudinal studies (see Butterworth et al. 1985; Dessein et al. 1988). We have been unable to demonstrate that the double monoclonal antibody EIA detects circulating native Sj26. The signal obtained in the assay could not be depleted by reaction of sera on glutathion+agarose col-

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IN

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a w'

0 1

2

3 COATING

4

5

6

ANTIBODY

FIG. 4. OD,, results of EIA with rSj26 (solid) and positive human serum pool at 15 dilution (hatched) using various coating antibodies and HRPO-MoAb250-7 as readout. Coating MoAbs used were MoAbSO-1 (l), Gib13 (IgM anti-PC) (2), and several control IgM MoAbs of other specificities (3-5). Results with BSA-coated wells are shown in lanes 6.

umns, a highly effective means of depleting Sj26 from worm homogenates or expressed Sj26 from recombinant E. coli homogenates (Smith et al. 1986, 1988). No evidence was obtained that the MoAbSO-1 and MoAb2507 bound to molecules of S. juponicum other than Sj26. There was weak cross-reactivity on the S. mansoni GST, Sm26, with MoAb250-7, and MoAbSO-1 bound to a GST of F. hepatica adult worms. When human GSTs were tested in the two-site EIA, they were unequivocally negative. The human GSTs tested included GSTl and GST2 which are present in human liver cells. Control Melbourne sera from individuals with no exposure to schistosomiasis were

consistently negative in the EIA as were a large number of Philippine infection sera. No positives have been detected to date in sera from individuals living in nonendemic areas for schistosomiasis. The two MoAbs were shown to be directed to epitopes on Sj26 that are different from each other and which are poorly immunogenic in infected patients as evidenced by competitive RIAs with human sera and labeled MoAbSO-1 or MoAb250-7. Cross-reactive anti-Sj26 antibodies can be detected in a variety of sera, but highly sensitive assays are required and the antibodies appear to be of low binding affinity and titer (Lightowlers and Mitchell 1989). The presence of antibodies to Sj26

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may lead to rapid clearance of the antigen from the circulation and thus decrease the likelihood of detection. They may also mask epitopes and block binding of the MoAbs in the EIA. The poor antibody responses in patients’ sera to Sj26, and in particular to the target epitopes of the MoAbs on Sj26, therefore must facilitate detection of any Sj26 in sera. The specificities of two IgM anti-Sj26 MoAbs are of some interest. The binding of the protein A-reactive MoAb250-7 depends on the presence of a free C-terminus of Sj26. This MoAb binds well to the Sj26P-galactosidase fusion protein, recombinant near-native Sj26, and to native GST isolated from adult S. juponicum worms. However, it does not bind to several “composite antigens” (fusion proteins) produced by pGEX vectors in E. coli in which additional sequences are present at the Cterminus. It also does not bind to the Sj26 product of the pGEX-1 vector which comprises Sj26 plus an additional 10 amino acids at the C-terminus. MoAb250-7 binds to a recombinant protein consisting of the mutine leukemia inhibitory factor with 8 amino acids from the C-terminus of Sj26 at its Cterminus (CT-LIF). As MoAb250-7 binds weakly to Sm26 (Table I), it is interesting to compare the sequences of Sj26 (GGGD HPPK) and Sm26 (GGGDAPPK) in this region. Presumably, the substitution of alanine for histidine significantly alters the epitope recognized by the MoAb and the binding is decreased. In contrast, MoAbSO1 binds to both Sj26 and C-terminally modified Sj26, including pGEX fusion proteins, but not to CT-LIF. The specificities of MoAbSO-1 and MoAb250-7 are clearly different despite indications of partial crosscompetition in competitive RIAs. Radiolabeled Sj26 could not be detected following incubation of positive sera with solid-phase MoAb, radioiodination of the beads, elution and immunoprecipitation with sera from rabbits immunized with Sj26, or either of the MoAbs in solid-phase

ET AL.

prior to SDS-PAGE. The levels of the material in the circulation are presumably so low as to be undetectable in gel electrophoresis analyses. There was no detectable GST activity in positive or negative human sera, when assayed directly using as substrates I-chloro-2,4-dinitrobenzene and GSH (Habig et al. 1974). This may be an indication of the extremely low levels of GST in the circulation. It may also indicate that GST is unavailable for detection in this assay when it forms part of a complex. With the assumption that immunoreactive material detected in the Sj26 EIA with some schistosomiasis sera is indeed S. japonicum-derived Sj26, questions arise as to the precise origin of the Sj26 and the form in which it circulates. The GSTs appear to be variably expressed at the schistosome tegument surface (Taylor et al. 1988; Holy et al. 1989). Conceivably, GST is bound “enzymatically” at the surface of the tegument of the several life-cycle stages of schistosomes which occur in the mammalian host and the amount reflects the degree of ongoing damage (lipid peroxidation?) at the worm surface. To date we have been unable to demonstrate a rise in circulating Sj26 levels immediately following PZQ administration to S. juponicum-infected mice and rabbits using the Sj26 EIA. Sera from infected mice and rabbits are negative in the assay. Some PZQ-treated humans occasionally show a slight increase in EIA activity (W. U. Tiu and E. G. Garcia, unpublished observations). Since the GSTs are highly reactive enzymes, released Sj26 should disappear rapidly from the circulation after PZQmediated destruction of worms. These essentially negative results raise an interesting possibility to account for circulating Sj26 in some infected individuals resident in an endemic area and presumably constantly exposed to cercariae. If Sj26 is bound to altered tegumental lipids (e.g., Danielson et al. 1987) on resident, but more particularly incoming parasites, the entire complex may

MONOCLONAL

ANTIBODIES

be sloughed as a result of sustained immune attack and accelerated membrane turnover in individuals who are relatively resistant to reinfection. Thus, a positive signal in the EIA may be an indicator of acquired resistance or concomitant immunity. If the serum Sj26 is bound in a complex with released altered membrane lipids through substrate binding sites, several of the results obtained with the two site EIA are explained readily. First, our consistent failure to deplete signals by passing strongly positive sera over glutathione columns may be due to complex formation. Moreover, complexing in a macromolecular form may serve to markedly increase signals in the EIA leading to a gross overestimation of the amounts present when sera are compared with recombinant (monomeric?) Sj26 in the Sj26 EIA. This would than account for the great difficulty we have encountered in demonstrating serum Sj26 using Western blotting. Finally, this complexing with altered tegumental lipids in serum would account for positive signals obtained using the anti-PC monoclonal antibodies in solid phase in the EIA with antiSj26 monoclonal as the readout. The fact that anti-PC MoAbs which led to a positive signal were of the IgM and IgA, but not IgG, isotypes may be explained by multipoint binding. This is suggestive of peculiarities of detection based on a large complex circulating in serum. PC-containing antigens have been reported in schistosomes (Pery et al. 1979). Thus, we posulate that Sj26 is present in sera of some individuals with schistosomiasis who are continuously exposed to cercariae and circulates as a complex with altered parasite tegumental lipids via substrate binding sites. These lipids may be altered as a result of immunemediated lipid peroxidation, the aldehyde products of which are known to be substrates for GSTs (Danielson et al. 1987). Regardless of the precise origin and nature of circulating Sj26 in schistosomiasis patients, a potentially illuminating series of

IN SCHISTOSOMIASIS

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experiments will be the search for antibody specificities and isotypes directed to (surface) antigens of worms, preadults, and schistosomules in individuals with high levels of circulating Sj26. It is hoped that this will lead to the identification of differentially expressed immune responses and their target epitopes in a subset of individuals who may be relatively resistant to reinfection, i.e., those who have developed efficient acquired or concomitant immunity. Such information should be relevant to molecular vaccine development in schistosomiasis japonica. ACKNOWLEDGMENTS This work was supported by the Australian National Health and Medical Research Council, the Rockefeller and Edna McConnell Clark Foundations, the LJNDP/ World BankiWHO Special Programme for Research and Training in Tropical Diseases, and the Australian International Development Assistance Bureau. We thank Rowena Almonte, Ruth Diasanta, Ben Ventura, and Edward Calica in Manila and Sorsogon, Philippines, and Anthea Ramos, Susan Wood, and Karen McLeod in Melbourne for excellent technical assistance. REFERENCES BERGQUIST, N. R. 1987. Prospects of immunological intervention in human schistosomiasis: Summary and conclusions. Acta Tropica 44, 108-117. BUTTERWORTH, A. E., CAPRON, M., CORDINGLEY, J. S., DALTON, P. R., DUNNE, D. W., KARIUKI, H. C., KIMANI, G., KOECH, D., MUGAMBI, M., OUMA, J. H., PRENTICE, M. A., RICHARDSON, B. A., ARAP SIONGKOK, T., K., STURROCK, R. F., AND TAYLOR, D. W. 1985. Immunity after treatment of human schistosomiasis mansoni. II. Identification of resistant individuals, and analysis of their immune responses. Transactions of the Royal Society

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