1 ~i- (4Q ( 1(N~c

Project duration: Effectiv r ing date 15 Sept. 1987-Effective conclusion (including a no cost extention) 31 Aug 1992.

Submitted to the Office of the Scieice Advisor U.S. Agency for international Development.

TITLE OF PROJECT Towars art Antigranuloma Vaccine in Schistosomiasis Mansoni: Molecular Cloning and Characterization of a Putatively Relevant Antigen.

PRINCIPAL INVESTIGATOR Dr. Joseph Hamburger, The Kuvin Centre for the Study of Infectious and Tropical Diseases, Hebrew univ ersity of Jerusalem, Israel.

COLLABORATOR

Dr. Davy K. Koech, Kenya Medical Research Institute (KEMRI).

Project number: C7-162.

Grant number: DPE-5544-G-SS-7019-00.

A.I.D. Grant Project Officer: Mr. David Muj'nex, Science Attache US Embassy, Tel-Aviv, Israel.

~IEC~ . F &i/i October 1993

199317NOV

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Table of Contents: Executive summary 3 Research objectives 4 Methods: 5

life cycle, animals and infection 5 Antigens 5 Antibodies 5 SDS-PAGE and Western blotting 6 Egg cultures 6 Determination of 35S-methionine 7 Determination of 3H-mannose incorporation 7 Extraction of RNA and separation of mRNA 8 In vitro translation in a cell-free translation system 8 Inmunoprecipitation 8 N-terminal amino acid sequencing 9 Deglycosilation 9 Cleavage of MEG polypeptide 10 Construction of a cDNA library 10 Oligonucleotide synthesis 10 Nucleotide sequencing 11

Resultr" 11 Characterization of MEG-related reagents 11 Proteins and glycoproteins synthesis and secretion 11 De novo synthesis of MEG-related constituents

in egg cultures 1 2 Synthesis of MEG-related constituents in a cell-free

translation system 1 3 N-terminal aminoacid sequencing and synthesis

of mixed nucleotides 1 3 Deglycosilation of MEG 14 cleavage of MEG polypeptide 14

Impact 15 Project Activities/Output 16 Project Productivity 16 Future Work 17 Literature Cited 17 Figures and tables 19

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Executive summary: Antigens secreted from eggs of the bilharzia parasite Schistosoma mansoni

induce a form of an immunological hypersensitivity manifested by the accurn.mation of typical

inflammatory cells around eggs which became entrapped in the host's tissues. This "granulomatous

rsponse" (GR) which leads to the major pathology in the desease becomes spontaneously smaller during

chronic stages of the disease. It is therefore thought that artifitial induction of such reduced response may

act as an anti-granuloma vaccine for reducing the pathology. The induction of reduced GR may also cause

a reduction in spread of the disease since the intensity of GR is directly correlated with the rate of egg

excretion to the environment. MEG, a major egg gl)coprotein of S. mansoni was previously purified

and shown by us to be a granuloma-inducing antigen. The purpose of the present project was to prove the

feasibility of cloning in bacteria of the gene that encodes the synthesis of MEG's protein moiety, and to

proceed accordingly by cloning and synthesis of this gene product in bacteria in order to enable

quantitative production for further structural and functional analyses. We first demonstrated by using

specific antibodies that MEG and/or related molecules are synthesized in eggs maintained in culture and ir

a cell-free system in which messenger RNA (mRNA) from eggs served as a template for direct synthesis

of eg proteins. The results thus ascertained that stable and functional MEG-specific mRNA is present in

S. mazsoni eggs, and that it can be harvested from them without loss of function. Analysis of the

molecular weights of the newly synthesyzed MEG-related molecules and the presence or absence of a

carbohydrate component on them suggested that the original gene product is split before carbohydrates are

added to form MEG and that subsequent cleavage occures for its secretion. mRNA from S. mansoni

eggs was subsequently used for preparing complementary DNA (cDNA) which, in turn, was introduced

into bacteria. Anti-MEG antibodies servrd to identify clones in which MEG-specific genetic information

was expressed. Further corfirmation of the identity of selected clones is underway based on initial amino

acid sequence information which we derived directly from purified MEG. Large scale production of

MEG's protein in bacteria and its structural and functional analyses are now, for the first time, within

close reach. Our efforts twards an antigranuloina vaccine complement attempts twards an'i-infection

vaccine which, so far, provided only partial protection from infection in animals (including experiments

that were carried out in primates in Kenya). The tc-hnologies and concepts r'"this study were shared with

the collaborating team in Kenya by training and joint work. Long term agreements on collaboration

between our institutions were signed as a result of this and of other AID sponsored projects and future

vaccination trials are expected to be carried out in both our institutions.

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Research objectives: MEG, a major egg glycoprotein of Schistosonma mansani was previously isolated by us and shown to be a granuloma­inducing antigen. The purpose of the present study was to prove the feasibility of cloning the peptide moiety of MEG and, in case such feasibility is proven, to proceed by cloning of the corresponding gene in bacteria in order to enable quantitative production for further structural analyses of this molecule and for examining its putative activity in induction of granulomatous response and its down regulation.

Schistosomiasis is a widespread disease in mauy developing countries including Kenya. Important water development projects are associated with the spread of s'chistosomiasis. These include projects for maintaining water constancy for drought and flood control, projects for generating hydroelectric power and projects for perennial irrigation. Such projects which are often associated with population ,elocation and with an increase in population densities have almost invariably been followed by an inc:eased spread of schistosomiasis, particularely among chieldren. Effective anti-schistosomal drugs are now available for community-based morbidity control of schistosomiasis, a strategy adopted by the World Health organization (1). This strategy, however, is confronted with the inevitable reinfection of treated subjects , the high cost of drugs, screening and drug delivery and its low sustainability (2). There is therefore a special interest in developing a vaccination approach for schistosomiasis control. Numerous anti-infection vaccination experiments were and still are being conducted in various laboratories (3). Candidate antigens have been identified and the coiresponding genes cloned in bacteria for quantitative production of the relevant antigens. However, vaccination experiments conducted in rodents and in primates so far yielded only partial protection . The notion of an antigranuloma vaccine is based on information which was accumulated prior to and after the start of our project on the granulomatous response (GR) and its modulation, which is briefly as follows: Antigens excreted from eggs entrapped in the host's tissues evoke the GR, which is a manifestation of cellular an humoral reactions under the control of discrete subpopulation, of T-cells (4,5). The GR has been implicated as a major factor in pathogenesis of schistosomiasis in animal models and in humans. During chronic stages of the disease the GR undergoes a process of down-regulation, known as spontaneous modulation (6) which is thought to provide the regulatory process required for the arrest of massive fibrosis and serious disease that can follow uncontrolled GR. Consequently the possibility has been raised that artificial induction of modulation may have the characteristics of an anti-granuloma vaccine (6). Such artificial modulation was indeed shown to be possible in experimental schistosomiasis (7). more recently, it 4as been shown that the size of the circurnoval granluloma is directly correlated with the rate of egg excretion (8). Therefore, efforts towards anti-granuloma vaccine, which complement efforts towards anti­infection vaccine, may result in a dual benefit of both reduced morbidity

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and reduced transmission. Studies twards the identification of granuloma inducing antigens started about 20 years ago by demonstrating that a soluble extract of S. mansoni eggs (SEA) induce GR. SEA was subsequently fractionated and the granuloma-inducing activity was located in its glycoprotein fraction. We were involved in the purification and characterization of two granuloma-inducing glycoproteins from S. mansoni ova, namely MSA1 (9) and MEG(10). Other investigators fractionated SEA and identified fractions which activate lymphocytes for in vitro release of lymphokines typical of GR (11,12). Our studies with MEG, on the other hand, repeatedly demonstrated its GR-inducing activity in vivo (13). Since removal of the bulk of tb. protein moyety of MEG abolishes its GR-inducing activity we assumed that cloning of the peptide moiety of MEG should provide a candidate recombinant antigen for in vitro and in vivo (xperiments for elucidating the relation between structure of this molecule nd its activity in inducing GR and GR modulation. The cloning of MEG

polypeptide should provide the first recombinant polypeptide or a granuloma-inducing schistosomal antigen. In this regard the project is innovative. In December 1991 we activated a grant obtained from another source. This grant enabled repeated attempts towards preparing a cDNA library and eventually prerparation of such a library, its screening and identification of numerous clones expressing polypeptides reactive with specific anti-MEG antibodies. Completion of the work up to overexpression and initial experiments on the in vivo activity of recombinant MEG polypeptide is currently underway.

Methods: Life cycle, animals and infection : Outbred female albino mice were infected, when 6-7 weeks old, by subcutaneous injection of 350 S. mansoni (Egyptian strain) cercariae shed by Biomphalaria glabrata which were exposed to 10 miracidia/snail >5 weeks earlier. After 8-9 weeks of infection the mice were sacrificed and the intestines collected for harvesting of eggs to be used for preparation of antigens and for in-vitro cultures.

Antigens: Eggs were harvested from the intestines of infected mice. SEA was prepared and underwent lectin affinity chromatography and ion exchange chromatography steps as previously described (10) for the purification of MEG. In addition, a monoclonal antibody (Mab) anti-MEG termed KCSmeg 22-1 (see below) was employed for purification of MEG by immunoaffinity on CNBr Sepharose-4B column as described by the manufacturer (Parmacia, Upsala, Sweden). An average of 170ug MEG were purified from 10mg SEA.

Antibodies: These were required for identification of MEG, for its purification by immunoaffinity, for immunoprecipitation experiments and for screening cDNA libraries. (a) Rabbit anti-MEG (R anti-MEG) abtibodies

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were induced by injecting purified MEG adsorbed onto NCP. The membrane was dried and powdered by mechanical grinding. The powder was suspended in 0.3ml sterile saline, and injected intracutaneously at multiple sites along the axillae. A total of 90ug MEG were given in the first injection and 3 booster injections of 60ug each were given at 3 weeks' intervals. A titer of 1:5000 was obtained as determined by an enzyme-linked immunoserbent assay (ELISA) (14) employing purified MEG adsorbed onto the microtiter plate. The fractional antibody activity with peptide epitopes was determined after abolishing carhohydrate, epitopes by treatment with sodium metaperiodate (15). (b) Murine monoclonal (Mab) anti-MEG antibodies were prepared by a procedure previously described for the preparation of Mab against S. mansoni egg antigens (14). Preliminary screening was carried out by ELISA employing SEA, and srceening for hybridomas producing anti-MEG Mab was carried out by a Farr-type radioimmunoassay (9). Mab were prepared in ascitic fluids and concentrated by ammonium sulfate precipitation. Anti-MEG Mab KCSmeg 22-1 was employed in this study for affinity purification of MEG (see above).

SDS Polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting: SDS-PAGE was carried out according to Laemli (16) in 10% acrylamide gel slabs. Western blotting onto nitrocellulose membrane (NCP) (17,18) was followed by reaction with rabbit anti-MEG diluted 1:500, or a normal rabbit serum control. Antibody binding was detected by reaction with alkaline phosphatase-conjugated goat antibody to rabbit Fab (BioYeda, Reliovot, Israel) diluted 1:1000, and followed by a color reaction with naplityl phosphate disodium.

Eag cultures: We have worked out a new method for obtaining a large number of eggs from the intestines of infected mice. Sterile schistosome eggs are usually obtained from the liver becouse it can be handled sterilly from its removal onward. Intestines, which contain a rich normal bacterial flora, present a special problem. a) Preparation of sterile eggs from intestines of infected mice: The intestines were flushed with 0.2M NaCI to remove their contents, then cut open longitudinally and the mucosa scraped out with a glass slide. Following several rinses with sterile 0.2M NaCI containing 100units of Penicillin/ml and 100gg Streptomycin/ml the intestines were left overnight at room temperature, and subsequently washed 10 times with the sterile NaCI-antibiotics solution. Sterile conditions were maintained throughout the homogenization trypsinization and sieving steps (10). Finally, the eggs were washed 10 times with sterile NaCI­antibiotics solution, and twice with culture medium (see below), by repeated centrifugations at 1000rpm for 3 min each. Intestines from 25 infected mice yielded up to 600,000 sterile ova. Of the mature eggs (60 to

70% of the total) 10 to 25% exhibited miracidial movement or hatcing within 15min after exposure to hypotonic conditions. (b) CulturC conditions and sterility checks: The culture medium consisted of MEM Eagle's without methionine (Biological Industries, Kibbutz Beit Haemek, Israel), supplemented with insulin (0.2u/ml), oxalacetic acid (150ug/mi), glutamine (29.2ug/ml), penicillin (100u/ml) and streptomycin (100gg/ml). Sterile ova (100,000-300,000) were suspended in culture medium (2-8ml) in a sterile plastic test tube (Falcon NO. 2057, Becton Dickenson Co., NJ, USA), and were incubated at 37 0C containing 5% CO 2 while rocking. Aliquotes (0.1ml each) of eggs suspended in culture medium were taken at the start and at the end of the incubatin period for sterility checks on nutrient agar plates. For metabolic labelling of egg proteins, 35S-methionine (Amersham , UK) at 100 -500uCi was added to the culture.

Determination of 35 S-Methionine incorporation into egg proteins in culture Aliquotes of e6 gs in culture fluid were taken at time intervals, and incorporation of 35S- Methionine determined. Total radioactivity retained in egg proteins was determined after washing the egg twice with PBS, once with 5% TCA and twice with Methanol. This was followed by overnight incubation of the eggs with 0.3ml Soluene-100 (Packard Industries Co., Ill, USA) at 500C in the dark for solubilization. Finally glacial acetic acid (6gl) was added, and the treated egg pellet subjected to scintillation counting (Minaxy Beta, Tricarb 4000 Series, Packard instiuments Co., Ill, USA). Incorporated of radioactive methionine into secreted polypeptides was determined by absorbing aliquoted culture supernatants onto NCP, then washing with PBS, drying and counting. In order to determine residual secretion of labeled proteins the eggs were initially incubated for 24 hours in the presence of radioactive methionine, then washed thoroghly and incubated for another 24 hours with fresh medium without radioactive methionine. Total radioactivity incorporated into secreted proteins was determined directly after adsorbing supernatant aliquotes onto lcm2 filter paper squares. The filter paper squares were boiled in 10% TCA for 20min, then washed inl00% ethanol, dried and counted for determining TCA precipitable counts. For det,.rmining incorporation of radioactive methionine into SEA, the eggs taken from the culture were thoroughly washed in PBS to remove external radioactivity, then suspended in PBS, ground and ultracentrifuged as previusly described (10). Radioactivity was determined in aliquotes of the metabolicaly labeled SEA, before and after precipitation in TCA as described above.

Determination of incorporation of H3.-mannose into egg glycoconiugates in culture: Ova (4.5x10 5 ) were cultured in 8ml DMEM minus glucose and supplemented with 100gg/ml Streptomycine and 100u/ml Penicillin, 292Ag L-glutamine, 0.2u/ml insulin and 250gg/ml oxalacetic acid. D 2 3 H-mannose (New England Nuclear, specific activity 30 ci/mMol, lmci/ml). Eggs were

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maintained in 5ml of supplemented medium plus Iml 3H-Mannose in a 20ml tissue culture flask at 37 0C in a 5% C0 2 atmosphere. Following 48h incubation egg viability was 23% and sterility was proven by innoculating supernatant samples on proteose peptone agar plates. Culture sopernatants were separated from eggs and SEA was prepared from the cultured eggs. Radioactivity was measured in SEA and in the culture medium by adsorbing aliquotes on NCP and subjecting the membranes to scintillation counting.

Extraction of RNA and isolation of poly(A)+ RNA: Total RNA was required initially for cell free in vitro translation experiments. Later on isolation of poly(A)+ RNA from total RNA was required for constructing cDNA libraries. Total RNA was extracted from S. mansoni ova by a variety of methods including the guadinium isothiocyanate (GIT) method of Chergwin et al. (19), the method of Chomczynski and Sacci (20) which combines guanidinium isothiocyanate with phenol/chloroform, and a method recommended for extracting RNA from eggs and embrios. A commercial kit for RNA extrtaction (Invitrogene) was also employed for this purpose. Poly(A)+ RNA was isolated by Oligo(dT) Cellulose (Pharmacia, or Promega) according to the manufacturers' instructions.

In-vitro translation in a cell-free system: RNA was extracted from eggs by the guanidinium isothiocyanate (GIT) method (19) after grinding 500,000 to 900,000 eggs for 15 min. at room temperature in GIT buffer (4M GIT, 25mM sodium acetate pH 6.8, and 0.83% beta-mercaptoethanol), by a glass to glass homogenizer (TenBroek homogenizer, Weaton, USA). Translation of the RNA (1 or 2ug) was carried out in a reticulocytes cell-free translation system (New England Nuclear, USA) according to the manufacturer's instructions.

Immunoprecipitation: This was required for identification of MEG-related constituents in metabolically labeled SEA and in cell-free translation products. (a) Identification of metabolically labeled MEG-related constituents in egg culture: NRS (100g.l) was first bound to Protein-A Sepharose as described by the manufacturer (Pharmacia, Upsala, Sweden). Sepharose-bound NRS was mixed with 800ul metabolicaly labeled SEA from a 24hr culture, incubated for 4hr at 370C while rocking. Aliquotes of 200g.l labeled and pretreated SEA were mixed with 20iti rabbit anti-MEG (or NRS or PBS as controls), and incubated for 4hr at room temperature. Complexes were removed after addition of 2401.i Protein-A (50% in precipitation buffer ) and incubation for 40min at room temperature while rocking, then processed and run by SDS-PAGE as usual. (b) Identification of MEG-related cell-free translation products: Translation products in 300PIl immunoprecipitation buffer were incubated with 2111 NRS for 10min at room temperature. Protein-A sepharose (40pl) was subsequently added and incubation carried out ay 40C for 30min while rocking. The supernatant was

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then treated with Rabbit anti-MEG at final dilution of 1:150, by overnight incubation at 40C. A control tube was incubated with NRS under similar conditions. Complexes were trapped by incubation with Protein-A Sepharose (80g1) for 30min at 40C. Immunoprecipitates were resolved by SDS-PAGE.

N-terminal amino acid sequencing of MEG: This was undertaken in order to enable construction of relevant oligonucleotides for further characterization of immunoselected MEG-related clones from a cDNA library, and for sequence information to help elucidate the correct reading frame of cloned MEG-polypeptide gene. Affinity purified MEG was subjected to amino acid sequencing by the Gas Phase Microsequencer 470-A (Applied Biosystems).

Deglycosilation of MEG; Attempts at deglycosilation of MEG were undertaken in order to enable a more accurate and extended amino acid sequencing and a higher antibody response to peptide epitopes of MEG. Deglycosilation was also necessary for facilitating digestion of MEG by proteolytic enzymes for peptide mapping and extended amino-acid sequencing. (a) Deglycosilation by trifluoromethyl sulfonic acid (TFMS) a chemical known to be a powerful deglycosilating agent (21). Two procedures (21,22) were employed and fetuin, a highly glycosilated glycoprotein as well as a crude-SEA glycoprotein fraction were used as control. In both cases the reaction was carried out in special vials (V-vials, Weaton) and TFMS mixed with Anisole was employed for the reaction under Nitrogen atmosphere, at 0oc. The extraction methods employed in the two procedures we used were somewhat different. The products were run by SDS-PAGE in 10% acrylamide gels for determinind alterations in the electrophoretic migration following TFMS treatment. (b) Deglycosilation by Endoglycosidase H (Endo­P-N-acetylglucosaminidase=H-Endo H). Endo H cleaves N-linked oligosaccarides from a glycoprotein leaving an N-acetylglucosamine residue attached to the polypeptide. There are some structural specificities which determine the susceptibility of an N-linked oligosaccharide to cleavage byEndo H (23). The enzymatic reactions were carried out according the instructions of the manufacturer (Boehringer). (c) Deglycosilation by Glycopeptidase F (PNGaseF). PNGase F cleaves an intact oligosaccharide from a native or a denatured glycoprotein. The cleavage occures between the innermost residue of N,N'-diacetylchitobiose and the aspargine to whoich the oligosaccharide is linked. It readily cleaves most high mannose oligosaccarides, hybrid oligosaccharides and biantennary, triantennary or tetra antennary complex oligosaccharides. The substrate specificity of PNGase F has been described in details (23). Initial experiments were carried out using the PNGase F manufactured by Boehringer at conditions recommended by the manufacturer, or at conditions described in the original paper of Haraguchi et al. (24). Fetuin and an N-glycopeptide 13­galactosidase-labeled, supplied by Boehringer as a positive control for

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PNGase F activity, were also subjected to digestion under similar conditions. Subsequent attempts were carried out by employing PNGase F from New England Biolabs. Following exposure to PNGase F the tested glycoproteins were run by SDS-PAGE in search of altered electrophoretic migration. The effect of PNGase F (Boehringer) on the control glycopeptide (Boehringer) was tested by Concanavalin A separation and measuring the activity of the released P3-galactosidase in the fall-through fraction according to the manufacturer's instructions. We also attempted using PNGase F for Deglycosilation of MEG adsorbed onto NCP with the thought of separating the enzyme from the remaining polipeptide following the deglycosilation reaction (25).

Cleavage of MEG polypeptide: This work was intended for preparing cleavage products suitable for further amino acid sequencing of MEG. This was particularely important for enabling PCR amplification of MEG sequences. The "Probe-Design" (Promega) peptide separation system was employed for this purpose. It involves cleavage of the selected protein by CNBr/formic acid prior to separation of the resuting peptides by high resolution SDS-PAGE and their blotting onto PVDF for amino acid sequencing. Proteolytic cleavage of MEG was also attempted by employing Trypsin and V-8 by procedures based on the method of Cleveland (26).

Construction of pn S. marsoni egg (-DNA library: Numerous attempt to prepare a cDNA library in phage Xgtll were undertaken. Initially this was done by a classical oligo (dT) primed first strand sinthesis coupled with the use of RNAase H-DNA Polymerase I-mediated second strand synthesis (27). The cDNA underwent methylation catalyzed by EcoRI methylase and then a filling reaction. EcoRI linkers were then kinased and ligated to the protected and blunted ends cDNA. This construct was ligated to dephosphorilated Xgtll arms and the ligation product packaged into the phage. Screening was carried out by 32p- labeled single stranded cDNA. Subsequents attempts were carried out by employing the "Riboclone System" (Promega) for synthesis of cDNA. The following steps of adding adaptors and subsequent ligation and packaging in vitro of insert DNA into lambda vector were also carried out using sutaible kits (Promega). following introduction of the recombinant phage into E. coli Y1090 the library was immunoscreened by employing rabbit anti MEG in a procedure adapted from Huynh et al. (28). Positive clones were purified for furthr analysis. Recombinant phage DNA was isolated by employing "Lambda Sorb" Phage adorbent (Promega).

Oligonucleotide synthesis: Based on amino acid sequence information obtained from the N-terminal end of MEG we synthesized mixed oligonucleotides taking into account codone usage of S. mansoni (29). The DNA Synthesizer 380B (Applied Biosystems) was used for this purpose. Two mixed oligonucleotides 19 mers were prepared since the sequence

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contained a serine for which there are four degenerate codons in position 3 after TC and two degenerate codons at position3 after AG. The degeneracyof the first oligonucleotide mix was 256, and that of the second was 128.

DNA sequencing : This is being done by dideoxy chain termination method of Sanger et al. (30) as modified for duble stranded sequencing (31). The "Sequenase" kit (United State Biochemical corporation) and universal lambda primers are being used. It has been shown that the use of a mixed sequence primer with degeneracy up to 512 yields a sequence indistinguishable in quality from that obtained with a specific unique primer (32) we currently attempt this approach too.

Results: Characterization of MEG-related reagents prepared: Figure 1 presents the results of SDS-PAGE of Immunoaffinity-purified MEG (a), and of Western blot analysis of SEA by rabbit antiserum prepared against purified MEG (b). In each case a single band (occasionally a doublet) of about 70KDa was exhibited. Table 1 presents the effect of periodate oxidation of the antigen on its binding to Rabbit anti-MEG and (o Mab KCSmneg 22-1. It can be seer, that while treatment of the antigen with 0.1mM NalO 4 completely abolishes binding of KCSmeg22-1, a concentration 100 times higher of periodate leaves about 30% of the Ranti-MEG binding activity, thereby suggesting that, unlike in the case of the Mab, polyclonal Rabbit anti-MEG includes a proportion of about one third, of antibodies to peptide epitopes of MEG. This makes it a potentially suitable reagent not only for specific detection of metabolically labeled MEG, but also for detecting translation products and of corresponding clones in a cDNA library .

Protein and glycoprotein synthesis and secretion by eggs in culture: Eggs were incubated in a medium containing 3 5 S-methionine and total radioactivity incorporated into the eggs (fig. 2a), as well as radioactivity incorporated into proteins secreted from the eggs (fig. 2b) were determined in aliquotes taken at hourly intervals. It can be seen that incorporation of radioactive methionine into the eggs starts during the first hour, assumes maximal rate between the 1st and the 3rd hour, and starts to level off between the 3rd and the 4th hour. By comparison, secretion of proteins into the medium starts only during the second hour of incubation, it proceeds at a constant rate for about two hours and starts to level off between the 4th and the 5th hour after onset of incubation. Measurements of incorporation and secretion were also taken after 24hrs incubation in the presence of radioactive methionine (table 2, column A) indicating that labeled proteins continued to accumulate during this period and reached about 18 fold the leval attained at 4hrs (22xl0 6cpm as compared with 12.4xlO5cpm seen in fig. 2a). About 17% of the total incorporated

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radioactivity (3.7xlO6cpm out of 22xlO 6cpm) was associated with TCA precipitable radioactivity in SEA. TCA precipitable secreted radioactivity reached a level about 22 fold higher (7.3xlO5cpm) than that observed after 6hrs incubation (3.2xl0 4cpm as seen in fig.2b). After 24hrs incubation the eggs were thoroughly washed to remove spent medium and radioactive methionine, and incubated for another 24hrs with fresh medium containing unlabeled methionine. As seen in table 2 column B, total incorporated radioactivity and TCA precipitable radioactivity in SEA were reduced by about 11% and 16% respectively, while TCA precipitable radioactivity from the culture supernatant was reduced by about 65%. It therefore appears that the reduced amount of TCA precipitable radiolabeled constituents in the supernatant, observed between 24 and 48hrs in culture, is largely the result of a true reduction in secretion rather than the synthesis of a specially high proportion of unlabeled secretable constituents. Foilowing 24h incorporation of radioactive mannose it was seen that 10.6% of the radioactivity in culture was incorporated into the eggs. Of the incorporated radioactivity 13.4% was found in constituents maintained within the eggs, and 86.4% in secreted constituents. Secreted constituents labeled with radioactive mannose were run by SDS-PAGE and the results are presented below.

De novo synthesis of MEG-related constituents in egg culture: This was analysed by immunoprecipitation followed by SDS-PAGE. SEA metabolically labeled with 3 5S-methionine (about 3xlO5cpm) was iminunoprecipitated with Ranti-MEG. A similar quantity was reacted with NRS as a control. Antigen-antibody complexes were precipitated with Protein A-Sepharose. The radioactivity in the immunoprecipitate constituted 0.74% of the SEA radioactivity input. This value is close to the yield of MEG isolated biochemically (0.85% of total SEA). Aliquotes of the immunoprecipitate were separated by SDS-PAGE (fig. 3) and newly synthesizes proteins were identified by autoradiography. These included the 70KDa band characteristic of MEG and 3 additional major bands with molecular masses of about 43KDa, 50KDa and 90KDa. In order to determine which of these constituents are glycosilated we separated SEA glycoproteins from the metabolically labeled (35 S-methionine) SEA by chromatography on Con- A Sepharose and subjected the labeled SEA-glycoproteins to SDS-PAGE and autoradiography. The 90KDa and the 43KDa bands were not observed among newly synthesized SEA-glycoproteins (fig. 4) suggesting that it is non-glycosilated. We therefore consider possible that the molecular weight of the initial MEG polypeptide gene product is of about 90KDa and that it is split into units of about 43KDa before it undergoes glycosilation. Clues on the possible relation between the 70KDa and 50KDa glycoproteins emerge from our comparative analysis of SEA and of constituents screted from eggs in culture. Numerous constituents including the 70KDa molecule (MEG) appear among metabolically labeled (35 S-methionine) SEA constituents when run by SDS-PAGE (fig. 5a). By comparison, the 50KDa molecule is the

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major constituent, but not necessarely the only one, among egg constituents secreted to the culture medium, and no constituents with higher Mr can be found among the secreted constituents except fcr some aggregates at the top of the gel (fig 5b). We therefore assume that the 70KDa glycoprotein (MEG) is cleaved into a 50KDa product upon secretion from the egg. SDS-PAGE analysis of the culture supernatant following metabolic labelling with radioactive mannose (fig. 6) showed that a large proportion of the labeled material was at a wide range of high molecular constituents. Since such materials do not appear among the 35 S-metionine-labeled constituents secteted into the culture medium (fig. 5b) we assume that these are aggregated oligosaccharides. In addition to these aggregates a 50KDa constituent appeared :n the culture supernatent in a form of a doublet. This finding strengthens our assumption that MEG is secreted as a 50KDa molecule, probably after the 70KDa molecule undergoes cleavage. Furtheremore, it appears that the 50KDa constituents is perhaps the only glycoprotein secreted and that the rest of the secreted material is carbohydrate in nature. Since culture supernatant inhibited the binding of radioiodinated MEG to murine chronic infection serum (results not shown) we conclude that MEG-related constituents were present in the culture fluid and that the 50kDa glycoprotein may be a breakdown product of MEG. The structural and functional nature of the carbohydrates in the culture fluid remains unknown.

Synthesis of MEG-related constituents in a cell free translation system: In 3 separate experiments the 35 S-methionine incorpotation ratio (in the presence of schistosomal RNA and with no external RNA added) ranged between 3.33 and 5.87 with lgg RNA, and was about 1.4 times higher with 2ug of RNA. Table 3 presents the results of two experiments of in-vitro translation of S. mansoni egg RNA Labeled translation products (about 1.4xlO6cpm) were immunoprecipitated with Ranti-MEG. A similar aliquote of translation products was reacted with NRS as a control. Antigen-antibody complexes were precipiteted with Protein A-Sepharose. The radioactivity found in the precipitate (4xlO4cpm) constituted about 2.8% of the translation product radioactivity input. Aliquotes of immunoprecipitated translation products were run by SDS-PAGE alongside with untreated and NRS-treated controls. As seen in figure 7 translation products of schistosomal RNA (lane 2) exhibited multiple bands at a wide range of molecular weights. Most of these bands did not appear in poducts of a translation system to which no external RNA was added (lanel). Schistosomal translation products treated with NRS exhibited a weak nonspecific activity (lane 3) and those precipitated with Ranti-MEG exhibited multiple bands (lane 4). Some of these bands overlap with the background bands shown in lanel and their specificity therefore cannot be considered assured. The bands which seem to be definitely specific exhibit

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molecular masses of 92KDa, 50KDa, 40KDa and 25KDa are marked by arrows. The presence of a molecular species of about 90KDa is in line with other findings suggesting that the initial gene product of MEG polypeptide may present a molecular weight of about 90KDa. Whether the other molecular species are breakdown products or products of incomplete synthesis is unclear.

N-terminal amiuo acid sequencing of MEG and synthesis of mixed oligonucleotides: An amino acid sequence of the first 10 residues was obtained as follows: 1. Leu or Gly; 2. val; 3. Asn*; 3. Ser*; 5. Pro; 6. Thr*; 7.Glu; 8. Gly; 9. Pro; 10. Thr* (those marked by * exhibited lower yields of phenylthiohydantoin and are therefore presumed to be glycosilated). Two 19 mer oligonucleotides were synthesized on the basis of amino acid sequences of residues 2 through 10. The structure of these oligonucleotides is as follows: "MEG-a": GT(N)AA(T/C)TC(N)CC(A/T)AC(A/T)GA(A/G)G. (256 degeneracy). "MEG-b": GT(N)AA(T/C)AG(T/C)CC(A/T)AC(A/T)GA(A/G)G. (128 degeneracy).

Deglycosilation of MEG: Deglycosilation of 125 1-MEG by TFMS was repeated several times but it was shown that the electrophoretic migration of the treated MEG remained unchanged while similarely treated fetuin, which was employed as a control molecule for deglycosilation, exhibited about 30% reduction in molecular mass (results not shown). Repeated attempts to deglycosilate 125I-MEG by PNGase from Boehringer also gave negative results but the control molecules (fetuin and P-galactosidase-labeled glycopeptide) also gave negative results suggesting that the product was inactive. [This was confirmed by the manufacturer who later on started to add glycerol to the enzyme for better preserving its activity. Deglicosilation was attempted again towards the end of the reporting period]. In order to find out whether the failures to deglycosilate MEG by TFMS are to to the radioiodination of MEG we investigated the effect of TFMS treatment of SEA on its binding to monoclonal murine anti-MEG antibodies (directed to CHO epitopes), to rabbit anti-MEG (directed to both peptide and CHO epitopes) and to infected mouse serum which we have found (unpublished) to react with peptide and CHO epitopes of MEG as well as with other egg antigens. The results of this experiment are presented in Table 4 . It can be seen that TFMS completely abolishes binding of murine anti-MEG MAb, it abolishes more than 60% of the binding of Ranti-MEG and 60-70% of the binding of IMS. It can be therefore concluded that the antigenic reactivity of MEG is affected by TFMS but whether it affects its molecular mass is still unclear. More recently we have employed PNGase from another source (New England Biolabs). MEG was deglycosilated to leave a molecule with Mr of about 45KDa ( fig. 8). This result is in line with the suggestion that N­

15

glycosilation of a 40-45KDa MEG polipeptide results in mature 70KDa MEG. This work is still in progress in particular for enabling proteolytic cleavage of MEG polypeptode (see below). Cleavage of MEG polypeptide: This was intended in order to enable extended amino acid sequencing of MEG polypeptide. Preliminary attempts to cleave MEG chemically by CNBr resulted in disappearance of stainable MEG upon high resolution SDS-PAGE. We therefore assume that MEG polypeptide is cleaved into wery small peptides whose resolution may require HPLC. This work is still continuing. Attempts at proteolytic cleavage of MEG by employing trypsin and V-8 gave negative results. We assume that proteolytic enzymes may have difficulties in reaching the polypeptide due to its high glycosilation. It should be recalled in this context that even a harsh proteolytic treatment by pronase has a limited effect on the molecular size of a small proportion of MEG molecules (10).

Impact: The present study was designed to examine the possibility for eventual development of an antigranuloma vaccine and it represents efforts towards cloning of a candidate antigen. In this regard it is a basic research and its results were not expected to be, and are actually not immediately applicable for the management of schistosomiasis in the collaborating country and in other developing countries. However, the present study made definite progress towards preparation of a recombinant candidate antigen and towards its extensive employment in studies on molecular aspects of granuloma formation and its modulation. We expect this progress to lead to experiments in laboratory rodents within the next two years and to experiments in primates within the next 4 years.

The present study enabled sharing with our Kenyan counterparts the relevaut concepts and technologies which are a mainstay in modern parasitological research nowadays but at the time the project was started were still hardy applied in the collaborating institute. The collaboration enaL.led both our groups to critically examine conceps of collaborative development of new biotechnologies in the service of management of tropical diseases. The laboratory in the developing country was encuraged to upgrade its leadership in future collaborations by assuming a position of pricipal investigatorship. This approach was adopted in a subsequent research proposal supported by AID/CDR.

The notion of the relevance of the research to the developing country was discussed and improvements of the collaboration were suggested. The components of the collaboration we.re to include a desirable proportict, of the following components: Screening+treatment as part of the overall national disease control efforts, joint developments of modern biotechnologies and strengthening of research capabilities. Attempts towards preparing Mab to S. haenatobium and towards preparing

16

S. matobium RNA were included in the project in ordei to enable inclusion of the survey aspect in the project. Accessibility to a few thousants school chieldren within the framework of the present project enabled us not only to rollect S. haenatobiun eggs from their urine but also to provide treatment to these chieldren. The collaborating laboratory was provided with the filtration tools necessary for a large scale collection of eggs and has used it in other research projects. Training in the hybridoma technology and subsequent effort to prepare Mab to S. haernatobiun in KEMRI enabled purchasing equipment for this purpose and to examine the requiredlogistics. Thus, although Mab to this parasite were not prepared, these efforts helped establish the hybridoma technology in KEMRI. The project also prompted training of a Kenyan team member in our laboratory on molecular biology. Our connections with Kenya were considered by the WHO when inviting the principal investigator to participate in a workshop which took place in Kisumu Kenya as a facilitator for training Kenyan junior scientists in writing research proposals.

During the study period we strengthened our relations with KEMRI, with the Division of Vector Borne Diseases in the Public Health Laboratoies of the Ministry of Healt and with the International Primate Centre who provided assistance in carrying out the present project. should future experiments ii! primates become relevant we expect to carry them out in Kenya. The highlight of the strengthening of our relations with KEMRI is the signing in 1991 of an official agreement on collaboration between the Kuvin Centre and KEMRI.

Project Activities/outputs: Internal seminars on the the project were held both at the Kuvin Centre and at KEMRI. Some of the results were informally discussed with colleagues during international meetings (American Society of Tropical Medicine and Hygiene, Boston 1991, and the 3rd International Symposiumon on Schistosomiasis, Brasil 1991). Abstracts were sent to the international conference on schistosumiasis, Cairo1993, and to the 4th International symposium on schistosomiasis, Brazil1993. Since cloning was the main goal of this project a paper on cloning of MEG is pending the completion of this part of the work. Thi. work is now being continued with other support. A paper on: "Synthesis of MEG, a granuloma inducing egg antigen of Scistosoma mansoni , in egg cultures and in a cell­free translation system" is being prepared for publication. It represents the completion of the first part of the work which involved the elucidation of the feasibility of cloning MEG polypeptide. A publication on cloning of MEG polypeptide and initial functional analyses is expected pending completion of this part of the work. The project involved training of 2 Kenyan technologists, 1 Israeli graduate student and one Israeli Ph.D. student (partial training). Israeli technitians who participated also required partialtraining since the project required a wide range of technical knowhow.

17

Project productivity: The Proposed work involved the following items: preparation of Mab and polyclonal anti-MEG -,.ntibodies-accomplished but preparation of antibodies to S. haernatobium antigens was not accomplished; In- vitro synthesis of MEG in egg caltures-accomplished; Synthesis of MEG in a cell free translation system-accomplished; Construction of egg cDNA library, accomplished; Cloning of MEG-partially accomplished;Overexpressio. and characterization of immunobiological activities of recombinant MEG polypeptide- Unaccomplished.

The work involved required expertise in Parasitology, immunoche-nistry and molecular biology. There was no prior information on the molecular structue of the target molecule and S. mansoni cDNA libraries were a rare comodity. It is now realized that the success of the project was dependent on too many unknown technical factors and that the time, personnel and budgetary requirements were under estimated. Specific difficulties encountered during the project were the following. (1) Artificially induced antibodies to MEG were found to be reactive with CHO epitopes and repeated attempts were required before rabbit antisera with partial (20%­30%) anti-peptide activity were obtained. (2) We were unable to standardize the procedures for preparing undegraded RNA from S. ,nanscni ova. (3) We encountered many unsuccessful attempts for preparing a cDNA library, and a small cDNA library which was initially prepared was damaged beyond repair due to a prolonged refrigeration failure during the absence of the PI and the responsible team member. (4) Deglycosilati'n of MEG proved tc, be much more complicated than initially realized, (5) Personal problems of team members which resulted in a prolonged absence or required recruetment of replacements. (6) Problems encountered in preparation of Mab in KENYA included repeated bacterial and fungal contaminations of cell cultures, problems in supply of perishable biological materials purchased abroad, and high mortality of Balb/c mice during the prolonged immunization period.

Considering the difficulties encountered we believe the project was productive since the main aspects leading to cloning were accomplished and since important molecular aspects of MEG synthesis and secretion were elucidated. Most importantly, over expression of a recombinant granuloma inducing antigen and its use in studies on induction of GR and its modulation is for the first time within close reach.

Future work: This should include characterization of the gene encodingMEG polypeptide and its product, overexpression of recombinant MEG po!ypeptide, functional analyses in-vitro and in-vivo, identification of immunobiologically active epitopes, elucidation of spacial and temporal aspects of MEG synthesis in the egg and its secretion fron:, the egg, and elucidation of the diagnostic potential of recombinant MEG.

18

Literature cited: 1) W.H.O. (1985). Expert Committee Technical Report Series No. 728. 2) Gryseels, B. & Polderman, A.M. (1991). Parasitology today, 2:244. 3) Taylor, D.W. (1991). Experientia, 47:152. 4) Phillips, S.M. & Lammie, P.J. (1986). Parasitology today, 1:279. 5) Xu, Y., Macedonia, J., Sher, A., Pearce, E. & Cheever, A.W. (1991). Inf.

Immun., 59:2934. 6)Warren, K.S. (1972). Trans. R. Soc. Trop. Med. Hyg., 66:417. 7) Weinstock, J.V., Bloom, A.H. & Kassab, J.T. (1985). J. Immunol., 125:560. ') Doenhoff, M.J., Hassounah, 0., Murare, H., Bain, J. & Lucas, S. (1986).

Trans. Roy. Soc. Trop. Med. Hyg., 80:503. 9) Pelley R.P., Pelley J.R., Hamburger, J., Peters, P.A. & Warren, K.S. (1976),

J. Immunol., 117:1553. 10) Hamburger, J., Lustigman, S., Siongok, T.K.A., Ouma, J.H. & Mahmoud,

A.A.F. (1982). J. Immunol., 128:1864. 11) Harn, D.A., Danko, K., Quinn, J.J. & Stadecker, M.J. (1989). J. Immunol.,

142:2061. 12) Lucas, N.W. & Boros, D.L. (1991). Inf. Immun., 59:941. 13) Lustigman, S., Mahmoud, A.A.F. & Hamburger, J. (1985). J. Immunol.,

134:1961. 14) Hamburger, J. Weil, M., Anton, 4. and turetzky, T. (1989). Am. J. Trop.

Med. Hyg., 46: 605. 15) Woodward, M.P., Young, W.W.Jr. & Blodgood, R.A. !1985). J. Immunol.

Meth. , 78:143.

16) Laerali, U.K. (1970). Nature, 227:680. 17) Towbin, H., Staehelin,T. & Gordon J. (1979). Proc. Natl. Acad. Sci. USA,

76:4350. 18) Burnette, W. N. (1981). Analyt. Biochem., 112:195. 19) Chirgwin, J.M., Przybyla, A.E., McDonald, R.T. & Rutter, W.J. (1979).

Biochemistry, 18:5294. 20) Chomczynski, P. & Sacchi, N. (1987). Analyt biochem., 162:156. 21) Edge, A.S.B., Foltynek, C.R., Hof, L., Reichert, L.F. Jr. & Weber, P. (1981).

Analyt. Biochem., 118:131. 22)Weiss, J.B., Aronstein, W. & Strand, N. (1978). Exp. parasitol., 64:228. 23) Maiey, F., Trimble, R.B., Tarentino, A.L. & Plummmer, T.H.Jr. (1989).

Analyt. Bicchem., 180:195. 24) Haraguchi, T, Fisher, S., Olofsson, S., Endo, T., Groth, D., Tarentino, A.,

Borchelt, T.R., teplow, D., hood, L., Burlingame, A. Lycke, E., Kobata, A., & Prusiner, S.B. (1989). Arch. Biochem. Biophys., 274: 1.

25) Aebersold, R.H., Leavitt, J., Saavedra, R.A. & Hood, L.E. (1987). Proc. Nat. Acad. Sci, USA, 85:6970.

26) Cleveland, D.W., Fischer, S.G., Kirschner, M.W. & Laemmli, U.K. (1977) J. Biol. Chem., 252:1102.

27 Gubler, U. & Hoffman, B.J. (1983). Gene, 25: 823. 28) Huynh, T.V., Young, R.A. & Davis, R.W. (1985). In "DNA Cloning: A

Practical Approach" (Vol. 1). D.M. Glover Ed., IRC Press Limited, Oxford,

England, pp. 49-79. 29) Meadows, H.M. & Wilson, A.J.G. (1989). Mol. Biochem. Parasitol., 36:

291. 30) Sanger, F. Nicklen, S.F. & Coulson, A.R. (1977). Proc. Natl. Acad. Sci. USA,

74: 5463. 31) Hattori, M. & Sakaki Y. (1986). Analyt. Biochem., 52:232. 32) Javed, A.A. & Joshi, S. (1990). Biotechniques, 9:28.

Figures and tables: Figure 1: Purified MEG and anti-MEG antidi. a) Immunoaffinity purified

MEG. Arrow marksthe position of &66 KDa molecular weight marker. b) MEG immi.nodetected on a Western blot of SEA, by rabbit anti-MEG antibodies. 1- SEA Electv, iotted onto NPC and stained by amido black. 2-SEA electroblotted onto NCP and immunostained by Mab 22-1. 3- SEA electroblotted onto NCO and immunostained with normal mouse serum (control).

a b

M.rKd] 1 2 3• MArKd)

66-- 66-,

I.-IV

20

Figure 2: Incorporation of ILS-methionine into S. mansoni egg constituents in culture, a) Incorporation secreted constituents.

into eggs. b) incorporation into

a.

13

12 -

11

.0

.

x :2

U

9

8

7

6

5

4

3

2

0 1 2

TIME

3

(HOURS)

4

b.

i( 34 44­

o 2-I.­ 1­

3 H2 4 6

TI ME (HOURS)

21

Figure 3: Synthesis of MEG and related molecules in egg cuiture, Lanes 1 and 2- SEA metabolically labeled with 35 S-methionine. Lane 3-Metabolically labeled SEA immunoprecipitated by Rabbit anti-MEG. Lane 4-Normal rabbit serum used as control for immunoprecipitation.

2 3 4Mrkdl

66 ,'

45 -'

29-.'

eil

22

Figure 4: SDS-PAGE of SEA-glycoproteins separated from metabolically labeled SEA by Con-A-Sepharose 4B. Molecular weights of the major bands are marked.

Tr

-70 Kd

I -50 Kd

Figure 5: SDS-PAGE of 35S-methionine-labeled egg constituents. Lane 1- constituents in SEA. Lane 2- constituents secreted into the culture fluid. Constituents at 70KDa and 50KDa are marked.

1 2

70 Kd­"-50 Kd

U

23

Figure 6: SDS-PAGE of 3H-mannose-labeled constituents secreted from eg in culture.

50KDa "p

24

Figure 7: Identification of MEG-related constituents among products of cell free translation of S, mansoni RNA. Lane 1- RNA control (no external RNA added). Lane 2-Total translation products from S. mansoni RNA. Lanes 3,5- Translation products precipitated with normal rabbit serum. Lanes 4,5- Translation products immunoprecipitated with Rabbit anti-MEG antiserum.

I12 3 4 5 6

45 --­29u 46-a'.

25

Figure 8: Deglycosilation of MEG by PNGase. Lane 1 and 4- Molecular weight markers. Lane 2- Deglycosilated MEG. Lane 3- Untreated MEG.

45KDa..

26

Table 1: Ide ;:fk,*atiuo of the differential binding of anti-MEG antibodies to peptide and carbohydrate epitopes of MEG as determined by treatment of the antigen with periodate.

ELISA O,D, with periodate-treated SEA

Anti-MEG antibodies

R anti-MEG (lot 1)

Periodate concentration 0 0.1 1.095 1.061

1 0.779

(rM) 5

0.395 10

0.340

R anti-MEG (lot 2) 1.470 1.374 0.861 0.330 0.242

Mab KCSmeg22-1 0.721 0 0 0 0

Rabbir anti-BSA (negative control) 1.931 1.876 1.909 1.914 1.945

Table 2: Incorporation of 3 5S-methionine into S. mansoni egg constituents

A: 24h with label B: 24h with label 24h without label

No. of eggs 2.5x10 5 2.5x10 5

Total incorporetion 22x106 cpm 19.7x10 6 cpm

incorporation into SEA 6.5x106 cpm 5.Ox 106 cpm

Radioactivity precipitated by TCA from SEA 3.5x10 6 cpm 3.1x10 6 cpm

Radioactivity in culture supernatant (Sup.) at conditions "B" not applicable 10.4x10 6 cpm

Radioactivity precipitated from Sup. by TCA 7.3x105 cpm 2.6x 105 cpm

27

Table 3: Cell-free in vitro translation of total RNA from S. mansoni ova.

RNA 35S-methionine in TCA-Quantity insoluble material (cpm)

RNA source (1tg) at 0 time at 60 min

No RNA (Negative control) 15,631 32,987

Yeast RNA (Positive control) 2 17,323 178,855

S. mansoni egg RNA 1 16,396 57,170 product no. 1 2 14,197 76,310

S. mansoni egg RNA 1 14,210 83,411 product no. 2 2 15,856 103,701

Table 4: Effect of treatment of MEG by TFMS on the binding of anti-MEG antibodies as determined by ELISA.

Antibody ELISA O,D, with the following antisera dilution KCSmeg22-11 IMS 2 NMS 3 Ranti-MEG4 NRS 5

1:100 a: 0.085 a:1.309 a: 0.199 a: 0.237 a: 0.159 b: 1.716 b: over b: 0.307 b:0.613 b: 0.252

1:1000 a: 0.091 a: 0.669 a: 0.076 a: 0.076 a: 0.093 b: 1.761 b: 1.665 b: 0.097 b: 0.137 b: 0.084

I-Mab anti-MEG; 2- Infected mouse serum (16-20 weeks of infection);3-Normal mouse serum; 4-Rabbit anti-MEG; 5-Normal rabbit serum.

a: Tested with TFMS-treated MEG. b: Tested with untreated MEG.