Bulletin of the World Heaohk Organozaton, 57 (SuppL. J). 93-110 (1979)

Localization and chemical characterization ofPlasmodium knowlesi schizont antigens

J. A. DEANS' & S. COHEN2

The identification ofmalarial antigenis that induceprotective immunity couldprovide arational basisfor developing an effectiv-e antirnalarial vaccine as wiell as specific serodiag-nostic tests indicative of clinical immune status. Since protective inmmunity isprobably in-duced by stage-dependent rather than stage-independent antigens, the antigenic compo-sition of different stages of Plasmodiumn knowlesi has been compared, and a limitedchemtical characterization undertaken. This information should provide some insight intothe types of preparative procedure appropriate for the purification offunctionally im-portant malarial antigens.

The current resurgence of malaria brought aboutby the lack of implementation of existing malariaeradication measures, the reduced susceptibility ofmosquitos to insecdcides, and the appearance of drugresistant strains of the parasite, have stimulated inter-est in the development of alternative methods ofmalaria control, including immunization. The malariaparasite has a complex life-cycle with two majorstages of development in the vertebrate host and asporogonic stage in the insect vector. Immunizationtrials have been performed in a variety of experi-mental systems and all stages of the parasite have beenused as immunogens (I). These investigations haveestablished the stage specificity of immunity inducedby vaccination with different developmental stages ofthe parasite, indicating that the stage-specific. notstage-independent, antigens have an essential role ininducing protection. The type of immunogen usedand the virulence of the host-parasite system investi-gated both influence the degrec of protection af-forded by vaccination. Of the erythrocytic stage para-sites, merozoites have provided the most effectiveform of vaccine against Plasmodium know(esimalaria in the rhesus monkey, and P.falciparurn indouroucouli monkeys; in both these test systems, ir-radiated or killed parasitized cells gave weaker pro-tection against blood-stage challenge.A detailed knowledge of the antigenic composition

of erythlocytic parasites at different developmentalstages and information pertaining to specific antigendistribution on the parasite or parasitized erythrocyte

Research Assistant, Department of Chemical Pathology, Guy'sHospital Medical School, London, SE1 9RT, England.

I Professor of Chenucal Pathology, Guys's Hospital MedicalSchool, London, SEI 9RT, England.

should lead to a better understanding of why someparasite preparations are superior to others foi immu-nization. Numerous analyses of plasmodial antigenshave been made over the last 15 years; methods ofanalysis include immunofluorescence, parasite agglu-tination, immunoprecipitation, and sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis. Im-munofluorescence studies have demonstrated theexistence of antigens that are common to sporozoitesand exoerythrocytic and blood-stage schizonts; quan-titative antigenic differences between trophozoite-and schizont-stage parasites have also been suggestedby immunofluorescence. The schizont agglutinationtest and iesults of immunization experiments haveboth provided evidencc for stage-dependent antigens.Recently, in a ciossed immunoelectrophoretic analy-sis, the antigenic compositions of sequential erythro-cytic stages of P. knowles) were compared, and apreliminary localization of antigens on the schizont orschizont-infected red cell membrane was made bycell-fractionation (2).

In this report, further evidence is presented, fromlactoperoxidase catalysed iodination of schizonts, forthe localization of antigens on the surface of P. know-lesi schizont-infected red cells. A partial chemicalcharacterization of some of the antigens is alsopresented.

MATERIALS AND METHODS

AnimalsImported rhesus monkeys (Mocaca mulatta) of

either sex weighing 2-4 kg were caged in a room arti-ficially illuminated between 05h 00 and 17h 00.

3875 -93-

94 3. A. DEANS & S. COHEN

Parasites

A Malaysian strain of Plasmodium knowlesi para-site designatedW originally obtained from the Walter-Reed Army Institute of Research, Washington, DC,USA, was used (3).

Preparation oferythrocytic ghostsGhosts were prepared as previously described (2),

essentially by the method of Fairbanks et al. (4) whichfollows the principles of hypotonic lysis defined byDodge et al. (5).

Preparation of different parasite stages

Ring-, trophozoice-, and schizont-infected redcells were lysed with saponin (2), using a modificationof the method of Zuckerman et al. (6). Blood for thering-stage parasite preparation was collected when theparasitaemia was 50%; 99.80o of the parasites werering forms and 0.2qo schizonts. Blood containing tro-phozoite-stage parasites was collected at a parasit-aemia of 11%lo; 92%o of the parasites were trophozoites,7% ring forms, and I o schizonts. Blood for the prep-aration of schizonts was collected when the parasit-aemiawas 10-50%o, and parasites were predominantlyschizonts with 8 or more nuclei. Centrifugation (350 gfor 8 min at 25°C) gave a layer containing 90-95%yoschizont-infected cells situated below the leukocytesand overlying the erythrocytes. Merozoites were iso-lated by the cell sieve method of Dennis et al. (7). Themerozoites, which were eluted in Medium 199, werecooled rapidly to 4IC and stored at this temperaturefor a maximum period of 2 h, prior to harvesting bycentrifugation at 3000 g for 10 min at 4 'C. Merozoitepreparations contained 1.6-3.4% parasitized redcells.

Preparation ofschizont and schizont-infectedred cell membranes

Schizont-infected red cells (96%o schizonts, 3 o ringforms, I No trophozoites) were disrtupted using a Stan-stead Cell Disrupter at a pressure of 3500 kPa(SOOlbf/in2) (2); approximately 60%o of the schizont-infected red cells were disrupted, but the liberatedmerozoites remained intact. Intact schizonts andmerozoites were pelleted by centrifugation at 3000 gfor 10 min at 4°C. The supernatant, after centrifu-gation at 3000 g, contained membrane fragments,presumably a mixture of schizont and infected red cellmembranes; these were collected from the super-natant by centrifugation at 37 000 g for 45 min at4°C. The mixed preparation of schizont and infectedred cell membranes was resuspended in PBS andstored at -70°C; it will be referred to as schizontmembrane.

Lactoperoxidase-catalysed iodinationofschizont-infected red cells

Purified schizont-infected red cells were washedthree times in Medium 199 at 4 IC prior to labelling.The reaction mixture consisted of approximately2 x 106 schizonts suspended in 1.0 ml of Medium 199to which were added 18.5 kBq (500 PCi) of Na'251I,150 yg of lactoperoxidase, 10 mg of glucose, and 3 IUof glucose oxidase.b The reaction, which was a}lowedto progress for 5 min at 20 IC, was terminated by theaddition of 9 ml of cold Medium 199, 1 mmo!/litre inKI. The cells were harvested by centrifugation at450 g for 10 mmn at 4 'C, and washed three times inMedium 199, 1 mmol/litre in KI. Labelled cells wereextracted with Triton X-100 (as described below), andthe soluble antigen preparation was stored in aliquotsat -70 "C.

Solubilization ofsamplesfor crossedimmunoelectrophoresisWashed erythrocyte ghost and parasite prep-

arations were treated with Triton X-100 (final con-centration 40 g/litre in PBS) for 1 h at 20°C. Non-solubilized material was removed by centrifugation ina Beckman Microfuge B (at approximately 11 000 g)for 5 min at4 'C. Triton X-I00 extracts were stored inaliquots at -70 OC.

Protein estimationThe protein content of samples was estimated by

the method of Lowry et al. (8). To aid solubilizationof the preparations, 0.1 ml of 100 g/litre SDS was in-cluded in the assay.

Immune serumSera from 8 immune rhesus monkeys that had been

immunized with P. knowlesi strain W I merozoites inFreund's complete adjuvant (FCA) and challenged byblood infection were pooled, and the immuno-globulins purified by ammonium sulfate precipitationfollowed by ion exchange chromatography on DE 32.The purified immunoglobulins were concentrated toone-quarter of the original serum volume. This im-munoglobulin preparation agglutinated W I strainschizont-infected cells to a titre of 512. Details of im-munization challenge, and days of serum collectionare presented elsewhere (2).

Variant specific antiserumVariant specific schizont-infected red cell-aggluti-

° Na'251 (carrier-free) was obtained from The Radiochemica)Centre, Amcrsham, Bucks, England.

b From the Sigina Chemical Co., London, England

CHARACTERIZATION OF P. KNOWLES] SCHIZONT ANTIGENS 95

nating (SICA) antiserum was prepared by the methodof Butcher & Cohen (3). This serum agglutinatedW Istrain schizont-infected cells to a titre of 128.

Crossed immunoelectrophoresisCrossed immunoelectrophoresis was carried out in

1.3-mm thick gels of 10 g/litre (1%) agarosec inbarbital-glycine/Tris buffer (ionic strength 0.02, pH8.6) containing 10 g of Triton X-l00 per litre, on glassplates (50 mm x 50 mm x 1 mm) (2). Detergent wasincluded in the gel but not in the electrode buffers.Samples (containing 50 pg of protein) were subjectedto electrophoresis at 5 V/cm for 75 rnin in a water-cooled LKB Multiphor. An agarose strip containingantigen was retained on the plate and the agarose onthe remainder of the plate was replaced with anothergel, 1.1 mm thick, containing antibody (50 ,l/ml).Electrophoresis in the second direction was performedat2 V/cm for 12 h.The gels were pressed, washed, dried and stained

for protein with Coomassie brilliantblue as previouslydescribed (2). Staining for lipid was performed withSudan black (9).

Comparison ofantigens by crossedimmunoelectrophoresis

In order to determine whether antigens of identicalmobility from different sources (e.g., ring- and tco-phozoite-stage parasites) were identical, the twosamples were mixed before the first-dimension elec-trophoresis of the crossed immunoelectrophoresis. Ifthe area delimited by the precipitate in question in-creased in comparison with the individual crossed im-munoelectrophoresis profiles, then it was concludedthat this antigen was common to both preparations.All combinations of the parasite preparations wereexamined (2).

Crossed immuno-affinoelectrophoresisP. knowlesi antigens containing a-D-glucosidic, a-

D-mannosidic, or sterically related residues that hindto concanavalin A were identified by crossedimmuno-affinoelectrophoresis by the method of Bog-Hansen (10). The agarose gel and electrophoresisconditions were identical with those used for crossedimmunoelectrophoresis. An intermediate gel (7 mmwide) containing 100 pg of con A-Sepharose per cm2was cast adjacent to the agarose strip containing anti-gen; a 10-nmm strip of agarose separated the con A-Sepharose-containing gel from that containing anti-body. Some immunoglobulins have cathodic

c Electrophoresis grade (Lot No 6541) from ICN Pharma-ceuticals, Laboratory Impex Ltd, Lion Road, TwLckenham, London,England.

migration at pH 8.6 so that it was necessary to includea second intermediate (blank) gel to separate the im-mnunoprecipitation lines from the first intermediategel.

AutoradiographyX-ray film (Kodak RP X-Omat) was placed on the

gel side of the dried and stained plates. The film wasdeveloped after exposure for 2-5 weeks.

RESULTS

Crossed immunoelectrophoresis of merozoite anti-gens against individual rhesus sera resulted in very dif-ferent profiles, demonstrating the inter-animal vari-ation in antibody response to malarial antigens fol-lowing vaccination and challenge (2). The pooledserum obtained by miLxing sera from 8 animals con-tained antibodies directed against a wider variety ofantigens than those present in many individual anti-sera; it also contained a more representative sample ofthose antibodies commonly present in animals ren-dered immune by merozoire vaccinatuon and sub-sequently challenged.No immunoprecipitates were formed in a crossed

immunoelectrophoretic analysis of a Triton X-100 ex-tract of normal rhesus erythrocyte ghosts against im-mune rhesus IgG. Thus, all immunoprecipitatesformed when parasite preparations were analysed bycrossed immunoelectrophoresis against the same anti-serum were due to parasite antigens, and not to con-taminating normal red cell membrane components.The patterns obtained following crossed immuno-

electrophoresis ofTriton X-100 extracts ofP. knowlesi(Wl variant) ring, trophozoite and schizont parasitepreparations against immune rhesus serum are shownin Fig. IA-C. The I1 most prominent immunoprecipi-tates have been assigned numbers. A tentative num-bering of the immunoprecipitates was made on thebasis of their electrophoretic mobilhty and confir-mation of the numbering was obtained from crossedimmunoelectrophoresis profiles of the 3 combinationsof the 3 parasite preparations, which were mixed inequal proportions prior to electrophoresis in the firstdimension. Antigens 1, 2, 3, 4, 5, 7, 8, 10, and 11 arepresent at all stages of parasite maturation, from ringforms through to mature schizonts; they may there-fore be regarded as stage-independent antigens. Many-of these antigens constitute a constant proportion ofthe parasite material at all stages of development, butsome. namely I, 2, 4, 10, and I I are present at relative-ly different concentrations, depending on the degreeof maturation. The contributions made by antigens 2,4, 10, and 11 increase as the parasites develop from thering to the trophozoite stage, and the contribution of4

1. A. DEANS & S. COHEN

2,S

. . . .v.w .I

, I

.4

CA rFig. 1. Crossed immunoelectrophoresis patterns of Trrton X-100 extracts of erythrocytic P. knowlesi parasite preparations.A. Ring forms; B. trophozoltes; C. schizonts. Samples containing 50pg of protein were applied to each plate; the anode forthe first-dimension electrophoresis was on the left. Electrophoresis in the second dimension was conducted Into gels con-taining immrune rhesus IgG 150 MI/mI). Numbers were assigned to the 11 most prominent immunoprecipitates on the basis oftheir electrophoretic mobilities.

continues to increase as the parasites form schizonts.The transition between trophozoite and schizont isalso accompanied by an increase in the relative amountof antigen 1 present.

Antigens 6 and 9 are stage-specific, botb are absentfrom ring-stage parasites; antigen 9 is formed as theparasite develops from the ring to the trophozoitestage, and antigen 6 is restricted to segmented schiz-onts and merozoites (Fig. 1).By isolating merozoites and schizont membranes

from mature schizonts and performing separatecrossed irnmunoelectrophoretic analyses, it is possibleto discern which of the antigens are expressed on theparasite and which on the schizont and infected redcell membranes. The crossed immunoelectrophoresisprofiles of merozoite and schizont membrane prep-arations analysed against immune rhesus serum areshown in Fig. 2A and 2B. Antigens 1, 4, 5, 7, and 8 are

restricted to the merozoites; they are completely ab-sent from the schizont and infected red cell mem-branes. Antigens 2, 3, 6, 9, 10, and II are present inboth schizont membranes and isolated merozoites.Antigen 2 constitutes a major merozoite antigen, butits contribution to the schizont membrane is verysmall; its presence in the schizont membrane prep-aration may be due to slight contamination of thispreparation with merozoite proteins. The major andmost characteristic antigen of the schizont and in-fected red cell membranes is number 6; it is also foundin merozoites. Several malarial antigens exclusive tothe parasite have been demonstrated by this analysis,but schizont membrane specific antigens have not.

Malarial antigens were also localized on theschizont-infected red cell membrane by lactoperoxi-dase-catalysed iodination of intact washed schizonts.The iodinated cells were extracted with Triton X- 100

42 > , .

f

A B. (

Fig. 2. Crossed immunoelectrophoresis profiles of Triton X-100 extracts of P. knowlesi. A. merozoites; B. "schizont mem-branes". 50 ug of protein were applied to each plate; electrophoresis in the second dimension was conducted into agarosecontaining immune rhesus IgG (5O pI/mi).

96

.tai

I1%

CHARACTERIZATION OF P. KNOWLESI SCHIZONT ANTIGENS

and analysed by crossed immunoelectrophoresis; thedried and stained gel was then autoradiographed. Byautoradiography it was shown that antigens 3, 6, 9,and 10 were available for iodination on the surface ofschizont-infected red cells (Fig. 3). Thus, the onlyantigens present in the "schizont membrane" prep-aration that were not iodinated when intact schizont-

infected red cells were labelled were antigens 2 and 11.The proportion of 125I incorporated into protein

(compaled to lipid) was determined by trichloraceticacid precipitation of an aliquot of the Triton X-100extract ofiodinated schizonts. Approximately 50% ofthe label was associated with protein, labelling ofmembrane lipid accounted for the other 50%

J-s2.*

a0 x

c

9

3 s

A 8

Fig. 3. Crossed immunoelectrophoresis of schizont antigens following lactoperoxidase-cataiVsed 1251-iodination of intactschizonts. A. Autoradiograph B. The same plate after Coomassie brilliant blue staining. Precipitates 3, 6, 9, and 10 wereradioactive,

Lipid-containing antigens were identified by stain-ing the crossed immunoelectrophoresis plates withSudan black. Antigens 6 and 9 gave strong stainingreactions with Sudan black, whereas immunoprecipi-tates 3, 4, 10, and 11 gave faintly stained lines; the re-

II* a

A

maining precipitin lines did not react.The specific interactions of macromolecules with

plant lectins can be used to identify and characterizemacromolecules, provided that the binding specifici-ties of the lectins are known. Concanavalin A (con A)

Fig. 4. Crossed immuno-affinoelectrophoresis of schizont antigens and the reference pattern. A. Crossed immunoelectro-phoresis of 50 pg of schizont protein with a blank intermediate gel as reference for the crossed immuno-affinoelectro-phoresis experiment in B. B. Crossed immuno-affinoelectrophoresis of 50 pg of schizont protein with concanavalinA-Sepharose (100 pg/cm2) in the intermediate gel

I

4

t,n, S,IX-w\*~~~~~~

B C

97

I

98 1. A. DEANS & S. COHEN

Table 1. The antigenic composition of P. knowlesi blood stage parasites at different stages of maturity, showing thelocalization of antigens on the schizont membrane and the identification of con A-binding and lipid-containing antigens

Parasite preparationr

Antigen Schizont Con A Lipednumber PRing Trophozoite Schizont Merozorle membrone '1l-Iabelled C bindingd staining5

1I + + ++ +

2 ++ + +14 i++ 1+1

3~T+ + * + + 4

4 _ ++ + + - +

5 + 1 _

6 ?+ t_++ t T 41t

7 + + _

a + + _

9 + + - + + ++ ++

10 - --i-+ + + + + + - +

11 - 41~ ~ ~~~~-++ 4- + +

* P kriowesr parasit preparations were analysed by CIE against immune rhesus IgG immunoprecipitates are denoted by +, the area beneath the inirnunoprecipi-tate is also ndicated by +, + or + + +b immunoprecipiales formed in CIE analyses have bean ossigned numbers based on the eiectrophoretic mobility of the antigenc Antigens labelied when schizont-miecied red calk were subjected lo lacloperosdase-calalysed iodintion.d Antigens binding lo con A-Sepharose 45 in crossed immunoelectrophoresis* Antiwns containing lipid which slan wih Sudan black

(from jack beans) reacts specifically with carbo-hydrates and glycoproteins containing a-D-glucosidic,a-D-mannosidic, and sterically related residues. Byperforming a crossed immuno-affinoelectrophoreticanalysis of schizont antigens, with an intermediate gelcontaining con A, those antigens incorporating a-D-glucosidic and a-D-mannosidic residues were identi-fied. Fig. 4B shows the immunoprecipitation patternof schizont-antigens, analysed against immune rhesusmonkey IgG, when Sepharose-bound con Ad wasincluded in the intermediate gel. The correspondingimmunoprecipitation pattern formed when a blankintermediate gel was interposed between the antigensand antibodies is shown in Fig. 4A. Comparison ofFig. 4A and 4B reveals the con A binding property ofantigens 1, 2, and 10 (demonstrated by the absence ofthese immunoprecipitates in Fig. 4B).

Crossed immunoeIectrophoretic analyses of P.knowlesi WI antigen preparations against SICA anti-WI antiserum gave entirely negative results; no im-munoprecipitates were formed on any of the plates.A summary ofthe stage specificity, localization and

partial chemical characterization of the I1 malarialantigens, identified by their precipitation reactionwith immune rhesus monkey IgG, is presented inTable 1.

d Concanavaln A coupled to Sepharose was obtained from Phar-macia Fine Chenucals, Uppsala, Sweden.

DISCUSSION

The antigenic complexity of Triton X-100 extractsof erythrocytic P. knowkesi WI parasites is revealedby crossed immunoelectrophoresis. Triton X-100 ex-tracts of normal rhesus ghosts do not react with theimmune rhesus immunoglobulin and this confirmsthe parasitic nature ofall imunoprecipitates formed.The antiserum was obtained from functionallyimmune animals and must include antibodies thatbave a role in protective immunity. The importance ofimmune serum in controlling P. knowlesi infection inrhesus monkeys vaccinated with merozoites has beendemonstrated by passive transfer studies (11). How-ever, it is not known whether protective antigen(s) ofP. knowlesi precipitate in crossed immunoelectro-phoresis. A correlation between the appearance ofsome precipitins and protection has been claimed inmurine malaria (12).

In comparing the antigens present in different de-velopmental stages of the erythrocytic forms ofP. knowlesi, only the 11 most prominent immunopre-cipitates were assigned numbers. Several other weakerprecipitates (many of which are lost upon photo-graphic reproduction) were also present. Parasitecomponents not detected by the analysis include thosethat were nonimmunogenic in the serum donors usedand others that were present in the Triton-insoluble

CHARACTERIZATION OF P. KOVOWLESI SCHIZONlT ANTIGENS 9

residues (solubilization of which would requirestronger surfactants).The crossed immunoelectrophoretic analysis has

clearly demonstrated the existence of both shared andstage-specific P. knowlesi antigens during the eryth-rocytic developmental cycle. The former (shared anti-gens) comprise 1, 2, 3, 4, 5, 7, 8, 10 and 11; the latter,stage-specific antigens, are numbered 6 and 9. Wilsonet al. (13), using a classification ofP.falciparun anti-gens based on their differential heat susceptibility,have also demonstrated the existence of both stage-independent (Lb and RI antigens) and stage-depen-dent (La' and La2) antigens, which are present pre-domninantly in mature erythrocytic schizonts. Since itis probable that protective immunity is induced bystage-dependent rather than stage-independent anti-gens, it follows that of all the antugens identified, anti-gens 6 and 9 constitute the most likely candidates asprotective antigens. If antigen 6 was responsible forinducing protective immunity, then vacciniation withparasitized erythrocytes would only be effective if theparasites were at the schizont-stage of development.However, if antigen 9 constituted the protective anti-gen, vaccination with trophozoire-stage parasitizederythrocytes might be effective.The effectiveness of different parasite preparations

asvaccines is likely to be influenced by the localizationof antigens. Schizont and schizont-infected red cellmembrane preparations contained few antigens whencompared with whole schizont-infected red cells. The"schizont membrane" antigens (3, 6, 9, 10, and 11)were also expressed by the intracel'lular parasite. Sur-face labelling of schizonts revealed that the majorityof antigens present in a mixed preparation of schizontand schizont-infected red cell membranes wereexposed on the outer surface of the schizont-infectedred cell membrane, assuming that the increased per-meability of the parasitized red cell membrane (14)did not allow penetration of lactoperoxidase. Anti-gens 3, 6, 9, and 10 were all labelled when intactschizont-infected erythrocytes were subjected tolactoperoxidase catalysed iodination, whereas antigen11 was not. Antigen 11 may therefore be expressed onthe schizont membrane, or on the inner surface of theinfected red cell membrane; alternatively it may be ex-pressed on the outside of schizont-infected cells, butfailed to become labelled owing to a lack of availableiodinatable residues.

Significant labelling of lipid occurred whenschizont-infected cells were iodinated; approximatelyhalf of the '25I was associated with protein, and theother half was bound to lipid. By staining the crossedimrmunoelectrophoresis plates with Sudan black,lipid-containing antigens were identified. Immuno-precipitates 6 and 9 gave fairly strong stning re-actions with Sudan black, whereas 3, 4, i0, and 11gave weakly stained lines. With the exception ofantigen 4, all the lipid-containing antigens were pre-sent in the schizont membrane preparation. Interest-ingly. all of the antigens that were labelled with 1261when intact schizonts were iodinated contained lipid.Hence lipid peroxidation played an important part inthe identification of schizont-surface antigens.

Crossed immuno-affinoelectrophoresis revealedthat antigens 1, 2, and 10 bound to con A, and there-fore contained a-D-glucosidic, a-D-mannosidic, orsterically related residues. Of these antigens, onlyantigen 10 was detectable in the schizont membrane,confirming the finding of Shakespeare (personal com-munication, 1978) that schizont-infected cells havefewer binding sites for con A than normal erythro-cytes. The con A binding antigens (1, 2, and 10) werepresent in merozoites, antigen 2 constituting a majormerozoite antigen. Attempts to agglutinate mero-zoites with con A were unsuccessful, but some conA-ferritin binding was detected (IS). Hence, theseantigens probably represent intracellular merozoitecomponents or are associated with the inner layer ofthe cell coat.Two stage-dependent antigens (6 and 9) have been

demonstrated: both are expressed on the outer sur-face of infected red cells, contain lipid, and do notreact with con A. Antigens detected by haemaggluti-nation (SICA reaction) are also expressed on the out-side of infected erythrocytes and their amountincreases with the maturity of the intracellular para-site. Attempts to identify the SICA antigen by crossedimmunoelectrophoresis were unsuccessful; no pre-cipitin lines were formed when schizont antigens wereanalysed against SICA antiserum. The failure todemonstrate SICA antigens could indicate that SICAantibodies are nonprecipitating (although their actionas agglutinins makes this unlikely). Other possible ex-planations are that SICA antigens are insoluble inTriton X- 100, or that the detergent interferes with theprecipitation reaction.

ACKNOWLEDGEMENTSThis work was supported by the Medical Research Council, Londor., and the UNDP/World Bank/WHO Special

Programme for Research and Training in TropLcal Diseases. We thank Dr W. H. G. Richards of the Weilcome ResearchLaboratones, Dr A. VoLler of the Nuffield Laboratory of Comparative Medicine and Dr G. H. Mitchetl of Guy's Hospitalfor providing immune rhesus serum, and Dr P. J. Campbell of the Standards Processing Section, National Institute forBiological Standards and Control. for freeze drying some samples of vaccine. We thank Dr P. Shepherd for helpfuldiscussions and Sally West for excellent technical assistance.

99

100 J. A. DEANS & S. COHEN

RESUME

LOCALISATION ET CARACTERISATION CHiMIQUE DES ANILGENES PRtSENTSDANS LES SCHIZONrES DE PLASMODJUMKNOWLES

La composition antigEnique du parasite Plasmodiumknowlesa a W comparEe, i ses divers stades erythrocytaires,par 61ectrosynerWse effectute a pardr d'un pool d'antis6ruinde singes rh6sus imniuns. Onze antig6nes principaux ont etidentafies; 9 sont prcsents k tous les stades du parasite; les 2autres, absents dans les formes annulaires, apparaissent soiti I'un soit a l'autre des deux stades suivants. Des differencesont 6galement ete constatkes dans la quantite relative decertains de ces antigines selon le stade. Pour localiser lesanttg6nes, deux m6thodes-1e fractionnement sub-cellulaireet le marquage A l'iode- 125 par catalyse peroxydasique-ont&t6 utilis&es en conjonction ave: 1'61ectrosyn6rese. La

membrane du schizonte, ainsi que celle de l'6rythrocytecontenant des schizontes, contiennent 5 des 1 antig6nes duparasite r6pertories. Quatre de ceux-ci ne sont prEsents qu'ala surface ext6rieure de la membrane de 1'6rythrocyte infect&par des schizontes. Tous les antig6nes presents sur la mem-brane de schizonte, ainsi que celui qui est associk exclusive-ment aux formes du parasite intra-ceUulaires, contiennentun lipide. Trois antig6es liant la concanavaline A ont kttidentifies, tous pr6sents quel que soit le stade, mais un scule-ment d'entre eux a &t6 trouv6 dans les pr6parations Abase demembrane de schizonte.

REFERENCES

I COHlEN, S. & MITCHEI L, G. H. Current topics inmicrobiology and immunology, SO: 97-137 (1978).

2. DEANS, J. A. ST AL Parasitology, 77: 333-344 (1978).3. BUTCHER, G. A. & COHEN, S. Immunology, 23:

503-521 (1972).4. FAIRBANKS, G. ET AL Biochemistry, 10: 2606-2616

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Organization, 37: 431-436 (1967).7. DENNIS, E. D. ET AL. Parasitology, 71. 475-481 (1975).8. LOWRY, 0. H. ET AL Journal of biological chemistry,

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antigen-antibody precipitates in gels. In: Methods animmunology and immunochemistry. New York &London, Academic Press, 1971, vol. 3, pp. 298-300.

10. B0G-H%NSEN, T. C. Analytical biochemistry, 56:480-488 (1973).

11. BUTCHER, G. A. ET AL. Immunology, 34: 77-86(1978).12. ZUCKERMAN, A. Experimental parasitology, 42:

374-446 (1977).13. WILSON, R. J. M. ET AL International journal for

parasitology, 3: 511-520 (1973).14. NEAME, K. D. & HOMEWOOD, C. A. International

journal forparasitology, 5: 537-540(1975).15. BANN1STFR, L. H. ET AL Bulletin of the World Health

Organization, 55: 163-169 (1977).