Immunol. Cell Biol., 65 (Pt. 3) 231-239 (1987)

© Antigenic analysis of Strongyloides raff/infective larvae and adultworms

by Carolyn Northern and David 1. Grove

(From the Department of Medicine, Queen Elizabeth II Medical Centre, University of Western Australia,Nedlands, Western Australia 6009.)

(Submitted January 29, 1987. Accepted for publication March 16, 1987.)

Summar]'. The protein composition of Strongyloides raiti infective larvae and adult worms extractedsequentially in water, sodium deoxycholate and sodium dodecyl sulphate (SDS) and their excretory/secretoryproducts were analysed by both one- and two-dimensional SDS-polyacrylamide gel electrophoresis. Whilemany bands common to all preparations and both siages of the worm were seen, a number of bands uniqueto each stage and preparation were identified. Western biol analysis of these larval and adult preparationsfor reaction with IgG and igM antibodies in hyperimmune mouse sera revealed a large number of antigens.These data provide a framework for analysis of protective and diagnostically useful antigens.

INTRODUCTION

Very little is known about the antigenicnature of the nematode, Strongyloides ratti.Infective larvae (lL) migrate through thetissues while the fourth stage larvae and adultworms (AW) are located in the mucosa of thesmall intestine. There is a paucity of informa-tion on whether there is significant antigenicvariation between different developmentalstages of the parasite. Further, the roles ofdifferent antigens in the genesis of humoraland cell-mediated immune responses and inthe induction of specific protective immunityare ill-defined.

As a first step towards identifying theseantigens and defining their roles, we haveanalysed the protein composition of S. ratti.We have compared the protein profiles of thesomatic constituents of IL and AW sequen-tially solubilised with different agents and alsothe excretory/secretory (ES) products of both

Abbreviations used in this paper: AW, adult worm;APB, antigen preparation buffer; BLOTTO, bovine lactotransfer technique optimizer; DOC, sodium deoxycholate;ES, excretory/secretory; IL, infective larva; Mr, molecularweight; NEPHGE, non-equilibrium pH gradient gelelectrophoresis: PAA, polyacrylamide; PAGE, polyacryl-amide gel eiectrophoresis; PBS, phosphate-buffered saline;SDS, sodium dodecyl sulphate; TBS, tris-buffered saline;2DE, two-dimensiona! electrophoresis.

worms. Some investigators of parasite antigenprofiles have simply used water soluble prepa-rations (1). This has the disadvantage of notpermitting extraction of all the antigens,particularly the relatively insoluble cuticularcomponents. Other workers have addressedthis problem by using harsh extraction proce-dures with detergents such as SDS in order toobtain maximal amounts of parasite antigens(2). The difficulty with this method, however,is that so many proteins are recovered thatanalysis becomes more complex. In order tosimplify analysis, a number of workers haveused sequential two-step solubilisations withwater and sodium deoxycholate (DOC), orDOC and SDS as the solubilising agents withthe result that different sets of antigens wereobtained (3, 4, 5, 6). We have extended thistheme by employing a three-step procedure inwhich S. ralti antigens were extracted first withwater, then the residue was treated with DOCand the remaining pellet was solubilised withSDS. This sequence was chosen becauseextraction with the milder detergent, DOC,permits the preservation of antigen-antibodyinteractions, whereas SDS is a highly effectivesolubilising agent but may denature theproteins (7). Having prepared proteins by thesevarious methods, they were analysed by one-

232 CAROLYN NORTHERN AND DAVID I. GROVE

and two-dimensional SDS-polyacrylamide gelelectrophoresis (PAGE). We then confirmedthe antigenic nature of some of these proteinsby identifying antibodies against them inhyperimmune sera utilising the Western blottechnique.

MATERIALS AND METHODS

Animals and parasiteFemale Sprague-Daw ley rats, 150-200 g in weight, and

female C57B16/J mice, 6-8 weeks old, were supplied bythe Animal Resource Cenire, Murdoch, Western Australia.A homogenic sirain of S. ratti was maintained by serialpassage in these animals as described previously (8).

Preparation of parasites

IL were obtained from rat faecal cultures, washed threetimes by centrifugation in water at 500^ for 10 min, thenfrozen at -80'"-' in antigen preparation buffer (APB)20 mM Tris-HCl pH 8 0 (BDH Chemicals, Poole, U.K.),5 mM ethylenediaminetetraacetic acid (BDH Chemicals),2 mM phenylmethylsulfonyl fluoride (Sigma, St. Louis,U.S.A.) and 100 units/ml Aprotinin"* (Bayer Pharma-ceuticals, Botany, N.S.W., Australia.)

AW were obtained from rats 7 days after infection with10,000 IL (9). The small intestine was removed, slit longi-tudinally, rinsed, then incubated in phosphate-bufferedsaline (PBS) pH 7-4 for 30 min at 37 '̂ which allowed theparasiies to crawl out of the tissues. The worms were iso-lated from the residual fluid by gravity sedimentation,washed several times in PBS containing penicillin (Glaxo,Boronia, Vic, Australia) 100 uniis/m! and gentamicin(David Bull Laboratories, Mulgrave, Vie, Australia) 5

l. They were then frozen at -80'-' in APB.

Preparation of parasite proteins

Somatic proteins were prepared from worms that werethawed, washed and resuspended in fresh APB. They wereIhen homogenised in a Braun'* homogeniser; equalvolumes of worm suspension and glass beads, 0-45-0 50/tm in diameier, were placed in a bottle in a rolalingcylinder cooled with rapidly flowing CO2 for 2 min. Thehomogenate was allowed to stand overnight at 4''. Thesupernatant fluid was then centrifuged at 5,000 g for 30min at i'-. The supernaiant fluid was respun at 100,000 gfor 60 min at 4''. This supernatant fluid was filteredthrough a 0-23 nm Miltex GV low protein binding filter(Millipore Corp., Bedford, U,S,A.); this is designated as"water soluble fraction'. The two pellet preparations werecombined and resuspended in APB containing l^Io DOC(BDH Chemicals) and allowed to stand overnight at 4''.The material was then ceniHfuged and filtered as before.This supernatant fluid Is referred to as 'DOC-solublefraction'. The two pellets were again combined and theprocess was repealed in APB containing 1% SDS (BDHChemicals). The final supernatant fluid is designated"SDS-soluble fraction'. The remaining pellet was di,scarded.Each fraclion was dialysed against water at 4^ over 24 h.Protein concentration was measured by the method of

Hartree (10). In order to prepare ES proteins, living ILand AW were washed three limes in PBS containing anti-biotics as before, then resuspended in aniibiotic-free APB.Viable worms were (hen placed in tissue culture flasks andincubated for 24 h at 37^ in a 5% CO; atmosphere. Thesuspension of parasites was ihen centrifuged at 500 g for10 min at room temperature. The supernaiant fluid, knownas the "ES fraction', was dialysed and the protein concen-traiion measured. If necessary, the concentration of ESfraction was increased by freeze-drying.

SDS-PAGE

All preparations were analysed initially by one-dimensional SDS-PAGE using ihe discontinuous buffersystem described by Laemmli and Favre (II). In brief,samples containing 5 fig protein and molecular weightstandards (LMW kit, Pharmacia Fine Chemicals, Uppsala,Sweden) were prepared in sample buffer, 62-5 mM Tris-HCl pH 6-8, 4% w/v SDS, 5% v/v 2-mercaptoethanol(BDH Chemicals). 10% glycerol (BDH Chemicals) and0-001% bromophenol blue (Biorad, Richmond, California,U.S.A.). These were then heated in a water bath at KK)°for 5 min. Samples were centrifuged at ll,OOOg for 5 minand the supernatant fluids were loaded on to ihe gels.Samples were run under reducing conditions on a 10%polyacrylamide (PAA) resolving gel with a 3% PAAstacking gel, Geis were stained with silver as described byPoehling and Neuhoff (12) and photographed. Relativemolecular weighls (Mr) of sample potypeptides werecalculated from the mobility of molecular weight standardsco-elecirophoresed in a separate well of each gel. Mole-cular weight estimaies of 94 kDa or above are extra-polations and only serve as approximate guides.Densiiomeiric scanning of individual tracks wasperformed with an Ultroscan Laser Densitometer (LKB,Bromma, Sweden).

Two-dimensional electrophoresis (2DE)

2DE was performed using a modification of the methodof OTarrell et al. (13), Samples were prepared in lysisbuffer, 9-5 M urea (Biorad), I'^a Nonidet P40 (Sigma),2% Pharmalyte 3-10 (Pharmacia Fine Chemicals) and 5%2-mercaploethanol. The first dimension was separated bynon-equilibrium pH gradient ' get electrophoresis(NEPHGE) on a 0-7 mm thick slab gel as described byFerreira and Eichinger (14), The second dimension wasseparated by SDS-PAGE on a 1'5 mm thick 10% PAAgel. These gels were stained with silver.

Western blot analysis

Immune mouse sera were raised using a multipleinfection schedule. Mice were infected percuianeously with1,000 S. ratti filariform larvae every 2 weeks for 3 monlhsand bled 7 days after each infection. These sera were desig-nated hyperimmune mouse serum, pooled, and stored at- 80".

Proteins separated by SDS-PAGE were transferred tonitrocellulose paper (Biorad) as described by Towbin etal. (15). In this instance, prestained molecular weightstandards (Bethesda Research Laboratories, MD, U.S.A.)were used. The nitrocellulose was then blocked in bovinelacto transfer technique optimizer (BLOTTO), 5% w/v nonfat dry milk, 0-05% Tween 20,0-01% Antifoam A (Sigma),

ANTIGENIC ANALYSIS OF STRONGYLOIDES 233

in 20 mM Tris, 500 mM NaCI, pH 7-5 (16) for 30 minal room temperalure in order to saturate unused proteinbinding sites. These blots were incubated overnight at roomtemperalure with hyperimmune mouse sera diluted 1:50with BLOTTO. The nitrocellulose sheets were ihen washedwith ihree changes of BLOTTO over 1 h. Equal volumesof horseradish petoxidase-conjugated, affinity-purified,goal anti-mouse IgM (Cappel) and goat anti-mouse IgG(Biorad) were combined, diluted 1:500 wilh BLOFTO, thenapplied to nitrocellulose for 1 h at room temperature. Theblois were again washed with ihree changes of BLOTTOover 1 h, then rinsed brietTy in Tris-buffered Saline (TBS),20 mM Tris, 500 niM NaCI, pH 7-5. Immune complexeswere visualised after incubaiion of blots in TBS containing0'05% 4-chloro-l-naphthol (Biorad) and 0-015% H,O.for 20 min. The nitrocellulose was then rinsed in doubledistilled water and photographed.

RESULTSOne-dimensional electrophoresis

The SDS-PAGE profiles of IL water-, DOC-and SDS-soluble preparations and ES proteins

are illustrated in Fig. 1 and the accompanyingdensitometric tracings are shown in Fig. 2 and3. Most proteins had an Mr less than 100 kDa.There was a large number of bands commonto all preparations made from infective larvaebut there were some that were unique to eachpreparation. Three heavily staining bands withMr of 36, 51 and 61 kDa were common to thewater-, DOC- and SDS-soluble preparations aswell as to the ES products. Between 25 and 30major peaks were noted by densitometry ofeach of these preparations.

The SDS-PAGE profiles of AW water-,DOC- and SDS-soluble fractions and ESproteins are illustrated in Fig. 1 and the accom-panying densitometrie tracings are shown inFigs. 2 and 3. Most proteins had an Mr lessthan 100 kDa. There was a large number ofbands common to all preparations but some

Mr(kDa)

- 94

- 67

- 43

- 30

- 20

- 14.4

2 3 8

Fig. 1. SDS-PAGE of Strongyloides ratti IL and AW preparations, stained with silver, IL water-.soluble fraction (lane I),IL DOC-soluble fraction (lane 2), IL SDS-soluble fraclion (lane 3), IL ES products (lane 4), AW waier-soluble fraction(lane 5), AW DOC-soluble fraclion (latie 6), AW SDS-soluble fraction (lane 7), AW ES products (lane 8). Molecularweight standards are shown on the right (kDa).

234 CAROLYN NORTHERN AND DAVID L GROVE

30 43 67 94

MOBILITY

Fig. 2. Densitomctric tracings of SDS-PAGE separationsof waier-soluble fractions (upper panel) and DOC-solublefractions {lower panel). The solid lines represent tracingsof IL and the dotted lines indicate tracings of AW.Molecular weight standards are indicated by arrows (kDa).

SDS

E/S

Fig. 3. Densitomeiric tracings of SDS-PAGE separationsof SDS-soluble fractions (upper panel) and ES products{lower pane!). The solid lines represent iracings of IL andIhe dotted lines indicate tracings of AW, Molecular weightstandards are indicated by arrows {kDa),

distinctive bands were seen with each prepara-tion. Again, three heavily staining bands withMr of 36, 51 and 61 kDa were seen in all AWpreparations.

The stage-specificity of each preparationwas analysed by both subjective examinationof stained gels and by comparison of densito-metric tracings. With water-soluble prepara-tions, dominant bands with Mr of 13-5, 22,35 and approximately 88 and 94 kDa were seenin IL, while in AW dominant bands with Mrof 11-5, 21, 23, 26, 34, 37. 55, 74 kDa andapproximately 84 and 102 kDa were noted.

With DOC-soluble preparations dominantbands with Mr of 15-5, 17, 34, 47 kDa andapproximately 84, 94, 96 and 98 kDa wereobserved in IL, while in AW dominant bandswith Mr of 27, 28 and 56 kDa were found.With SDS-soluble preparations, dominantbands of Mr 17 and 29 kDa and approximately84, 94, % and 98 kDa were observed with IL,while no clear-cut dominant bands were seenin AW. The ES preparations are notable fortheir degree of similarity, yet stage-specificdominant bands with Mr of 30 and 37 kDaand approximately 94 and 102 kDa were notedin IL while bands of Mr 38 kDa and 15 kDawere noted in AW.

Two-dimensional electrophoresisGreater discrimination of these differences

was provided by 2DE. Large numbers of spotswere apparent in all preparations, but theyvaried in intensity. Comparison of IL and AWwater-soluble fractions (Fig. 4) show that morespots of lower Mr and pi were present in theAW fraction. In the DOC-soluble preparationscomparable heavily staining spots were seenwith both IL and AW fractions, but additionalheavily staining spots were seen in the AWfraction. With the SDS-soluble preparationstwo very intense spots were seen in the ILfraction, but a large number of additional wellstained spots were seen in the AW fraction(Fig. 5). The ES preparations of IL and AWshowed very similar profiles. The majorexceptions were two spots in the IL fractionand one further spot in the AW fraction(Fig. 5).

ANTIGENIC ANA[,YSIS OF STRONGYLOIDES 235

I kDa

-94

1

Fig. 4. Two-dimensional eleclrophoretic profiles of S. ratti IL (left-hand side) and AW (right-hand side) water-solublefraclions (upper panels) and DOC-soluble fractions (lower panels). Samples were analysed by NEPHGE in the firstdimension and SDS-PAGE in the second dimension. Acidic and basic regions of the gel are indicated. Molecular weightstandards are shown on the right (kDa). Brackets enclose dominant stage-specific spots.

Western blottingAnalysis of the larval and adult

preparations by Western blotting revealed alarge number of antigenic bands (Fig. 6). Thewater soluble IL preparation contained twostrongly staining bands; they were not seen inthe AW water-soluble fraction. The U. DOC-soluble preparation contained approximately13 darkly staining bands; some of theseseemed to correspond with moderately stain-ing bands in the AW DOC-soluble fractionwhile others were unique to the IL. The SDS-

soluble IL preparation revealed three majorsero-reactive components, none of which werevisible in the AW SDS-soluble preparation.The IL ES preparation contained two majorand a number of minor reactive bands, whilethe AW ES preparation contained more than adozen moderately staining bands. Comparisonof these bands between the stages revealedsome common and some stage-specific com-ponents. Thus, with all preparations bothshared and stage-specific antigens wereidentified.

236 CAROLYN NORTHERN AND DAVID L GROVE

I kDa I

- 9 4

- 9 4

- 6 '

Fig. S. Two-dimensional electrophorelic profiles of S. ratti IL (left-hand side) and AW {right-hand side) SDS-solublefractions (upper panels) and ES products (lower panels). Samples were analysed by NEPHGE in the first dimensionand SDS-PAGE in the second dimension. Acidic and basic regions of the gel are indicated. Molecular weight standardsare shown on the right {kDa), Brackets enclose dominant stage-specific spots.

DISCUSSION

We have shown that both the somaticextracts and the ES products of two develop-mental stages of S. ratfi are highly complex butthat both common and stage-specific proteinscould be readily demonstrated. Similar obser-vations have been made with a number ofother nematode parasites including Trichinellaspiralis (17), Angiostrongytus canlonensis (18),Nemalospiroides dubius (19) and Dipetalo-nema viteae (20). Furthermore, we haveshown, utilising sera from mice infected

repeatedly, that many of these compounds aresero-reactive. Some antigens appeared to becommon to both IL and AW but others werestage-specific. In general, the intensity ofserum antibody reactions appeared to begreater against IL than against AW antigens.This is not surprising, as many IL in pre-exposed mice may be destroyed during theirmigration through the tissues and fail to reachmaturation in the gut (21, 22). If antibodytitres against adult worms fall with time, asseems likely, then adult worm-specific anti-

ANTIGENIC ANALYSIS OF STRONGYLOIDES 237

Mr(kDa)

200-

97-6-

6 8 -

4 3 -

I

25-7-

18-4-

14-3-1 8

Fig. 6. Western blots of S. raiti IL and AW preparations with hyperimmiine mouse sera. It, water-soluble fraction{lane I). IL DOC-soluble fraclion (lane 2). IL SDS-soluble fraction {lane 3), AW water-soluble fraction {lane 4), AWDOC-soluble fraction {lane 5). AW SDS-soluble fraction {lane 6), IL ES products (lane 7), AW ES products (lane 8).Prestained-molecular weight standards are shown on the left {kDa).

bodies in the pool will be diluted by the laterserum specimens.

An analysis of the antigenie composition ofS. ratli is necessary for a number of reasons.Firstly, it is a prerequisite to explaining themechanisms of differences in the host responseto various stages of the parasite. Severalinvestigators have shown that there is a disso-ciation in the response of rats to infection withlarval and adult S. ratli (23, 24). Secondly,immunoassays provide a potent means for thediagnosis of strongyloidiasis. Most immuno-assays in the past have concentrated on the

detection of specific antibodies, but theseassays are not able to differentiate betweenpast and present infection (25). Assays for thedetection and measurement of Slrongyloidesantigen in the serum would be a majoradvance. The sensitivity and specificity of suchassays are likely to be enhanced if definedantigens are used or detected. Thirdly, thedevelopment of an effective vaccine would bea great advance in the control of strongyloi-diasis. This goal is more likely to be achievedonce antigens potent in the generation ofprotective immunity are identified and iso-

238 CAROLYN NORTHERN AND DAVID I. GROVE

laied. Such information would facilitate theproduction of such antigens by recombinantDNA techniques.

Those antigens most likely lo be of greatestrelevance in the generation of protectiveimmunity, in the stimulation of antibodiesmeasurable by immunodiagnostic tests, and inassays for Strongyloides antigen, will probablybe found in either the ES products or on thesurface components of the parasites. We havedescribed the antigenie profiles of the ESproducts of IL and AW and have shown thatdifferent antigens exist. We are in the processof identifying which of the somatic antigensare located on the cuticle of the worm bylabelling the surface with radioactive iodine.

The information that we have garnered thusfar provides a framework for further analysisof serological responses and assessment ofhost-protective immunity. Since it has beenshown previously that mice can be immunise'dwith the water-soluble preparation of 5. rattiIL (26), we plan to measure resistance to infec-tion in mice immunised with these variousfractions of IL and AW in order to ascertainwhich stage of the parasite is likely to be mostrewarding in the search for candidate vaccineantigens.

Acknowledgements. This study was supported by a gramfrom the National Health and Medical Research Councilof Australia.

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