Bulletin ofthe World Health Organization, 61(1): 105-112 (1983) ©) World Health Organization 1983

Plasmodium falciparum polypeptides released duringin vitro cultivation*

L. RODRIGUEZ DA SILVA,1 M. LOCHE, R. DAYAL, & L. H. PERRIN,3

-Synchronous cultures of Plasmodium falciparum were successively labelled with(3S)-methionine and both the supernatants and the pellets of infected red blood cells werecollected. The release of TCA-precipitable material in the culture supernatants was lowduring the development of ringforms and trophozoites, increased during schizogony, andwas maximum at the time of schizont rupture and merozoite reinvasion. Analysis of thesupernatants by SDS- PAGE and autoradiography showed that both polypeptidescommon to the various developmental stages of the parasite and schizont/merozoite-specific polypeptides were released. Polypeptides of relative molecular mass 140000,82000 and, to a lower degree, 41000 were present in high amounts in the culture super-natants. These polypeptides have been shown to be the target of monoclonal antibodiesthat are able to inhibit the growth of P. falciparum cultures, and may be involved inprotective immunity. The released polypeptides may also be used as target antigens inimmunodiagnostic tests aiming at the detection of malaria infection.

The sera of individuals with an acute Plasmodiumfalciparum infection are known to contain solublemalaria antigens (1, 2). These antigens have beendetected by double diffusion in gels using, asantisera, the sera of adult individuals who hadrepeatedly been exposed to P. falciparum infection(1, 2). Several malaria antigens have been recognizedand they have been classified on the basis of theirthermostability (3). Some of these antigens have alsobeen characterized by their relative molecular massand susceptibility to proteolytic enzymes (4). Otherstudies, using short-term in vitro cultures ofP. knowlesi and P.falciparum, showed that malariaantigens are released during the schizont develop-mental stage and/or at the time of schizont ruptureand merozoite invasion of new erythrocytes (5, 6).Some of the polypeptides released by the parasite inthe supernatant of short-term P. falciparum culturesin vitro, have the same immunological reactivity asantigens detected in the sera of infected individuals(5). The precise origin of the released antigens hasnot been elucidated.

* From the WHO Immunology Research and Training Centre,Geneva Blood Centre, Department of Medicine, Geneva UniversityHospital, 1211 Geneva 4, Switzerland.

' Assistant.2 Post-Doctoral Fellow.3Research Associate.

The aim of the present study was to determine andquantify the sequential release of P. falciparumantigens during the asexual development of theparasite and to identify at the molecular level theantigens released in the culture medium.

MATERIALS AND METHODS

Culture conditions and synchronization of theculture

P. falciparum (SGE1 strain) was adapted to invitro culture by using blood taken from a Europeanpatient who had a first acute P. falciparum infectionafter returning from Senegal. The adapted isolatewas propagated in group A red blood cells (RBC) bythe candle jar method (7). This isolate did notproduce gametocytes under the culture conditionsused.

Synchronized cultures were initiated and themultinucleated forms of the parasite were separatedfrom ring forms and normal RBCs by flotation inPhysiogel (8). Over 75%o of the cells floating inPhysiogel were parasitized RBCs. These cells weremixed in a ratio of 1 to 3 with fresh uninfectedRBCs. The reinvasion period was limited to 5 h,after which the remaining mature forms were lysedwith 5%/o mannitol (9). The procedure was repeated

425 -105-

L. RODRIGUEZ DA SILVA ET AL.

over a second replication cycle to increase synchronyand parasitaemia. In the experiment reported here(the experimental protocol was repeated three timeswith similar results), a pellet of 0.7 ml RBC with aparasitaemia of 2307o (93.7% ring forms) was dilutedin 28 ml of culture medium and divided in sevenPetri dishes (60 mm diameter). The culture mediumwas changed every 6 h and the course of theerythrocytic development was assessed micro-scopically by using Giemsa-stained thin smears.

Labelling conditions

The synchronized cultures were labelledsuccessively every 6 hours as follows: 0-6, 6-12,12-18, 18-24, 24-30, 30-36, and 36-42 hours.Successive cultures were washed once in methionine-free MEM (minimum essential medium), supple-mented with 10%7o human serum, 2 mmol of glutamineand 25 mmol of HEPES buffer, and resuspended in3.5 ml of the same medium containing 1.48 x 104 Bq(40 ACi) per ml of L-(35S)-methionine (specificactivity 3.84x 102 Bq (10.39 mCi)/ml). After 6 h oflabelling, the parasitized RBC were pelleted bycentrifugation at 1000 g for 10 min and then thepellets and the supernatants were treated separately.The pelleted cells were washed twice in coldphosphate-buffered saline (PBS), lysed by theaddition (to the last pellet) of 0.6 ml of TNE (Tris-NaCl-EDTA buffer, pH 8) containing 0.50%o ofNonidet P40 (NP40) and kept in aliquots at - 75 'C.The supernatants were centrifuged at 20 000 g for 30min at 4 'C and dialysed overnight at 4 'C againstPBS and stored at - 75 'C.

Analysis of the labelled material

Measurement of radioactivity associated withinfected RBCs and released in culture medium.Duplicate 10 Al aliquots of the various infected RBClysates and 100 pl aliquots of the supernatants wereincubated for 15 min at 37 'C in 0.5 ml of 1 mol/lNaOH containing 25 Al of hydrogen peroxide (30volumes) and precipitated with 3 ml of cold 257otrichloroacetic acid (TCA). The precipitates werefiltered on 0.2 A millipore filters and, after severalwashings with 5% TCA and alcohol, dissolved inReadysol scintillation fluid and counted in a Beck-man LS20 $-counter. The radioactivity associatedwith macromolecules in the infected RBCs andculture supernatants was expressed as a percentageof the total radioactivity (counts per minute (cpm) of(35S)-methionine added to individual cultures at thebeginning of each labelling).

Immunoprecipitation assay. Immunoprecipitationwas carried out as described by Kessler (10).Aliquots of culture supernatants containing TCA-

precipitated polypeptides (0.5 x 106 cpm) werebrought to a final concentration of 0.5/o NP40 and afinal volume of 400 Al by the addition of TNEbuffer. These samples were were incubated for 1 h atroom temperature with 10 Al of each individualserum. A volume of 50 Al of a 10o suspension offormalin-treated Staphylococcus aureus ("Pan-sorbin" from Behring) was added to the antigen-antibody mixture. After 15 min of incubation, themixture was centrifuged (2000 g for 10 min), and theprecipitates washed five times. The bound antigen-antibody complexes were eluted with 50 Al of sodiumdodecyl sulfate (SDS) polyacrylamide gel electro-phoresis (PAGE) sample buffer at 60 °C for 15 min.The pellets of labelled infected RBCs were solubi-lized in TNE buffer, pH 7.2, containing 0.5% NP40and then centrifuged at 20 000 g for 30 min at 4 'C.The supernatants containing soluble polypeptides(accounting for 90% of the measured cpm) wereused for immunoprecipitation, following the sameprocedure as for the culture supernatants. Three serafrom adults living in a malaria endemic area (inKenaba, the Gambia) and a serum from a Europeanwho had never suffered from malaria were used forthe immunoprecipitation assay.SDS-PAGE analysis and autoradiography.

Electrophoresis was carried out in 10% slab gel, asdescribed by Laemmli (11). All samples wereequilibrated in Tris-HCI-buffer, pH 6.8, containingiWo SDS and 2% mercaptoethanol. The gels werecalibrated with standard protein markers (PharmaciaFine Chemicals), fixed, stained with 0.1% Coomassieblue, dried, and autoradiographed on X-ray films(Kodak) for 5-10 days at - 75 'C.

RESULTS

A good synchrony of P.falciparum cultures wasachieved by using the methodology described (Fig. 1).The incorporation of (35S)-methionine intoparasitized RBCs varied during the course of theasexual cycle. As ring forms matured into tropho-zoites, the incorporation of (35S)-methionineincreased, reaching its highest level in mature tropho-zoites. A decline of incorporation was later observedin schizonts. The measure (cpm) of TCA-precipitablepolypeptides in the supernatants of cultures was usedto quantify the amount of parasite polypeptidesreleased into the supernatants. Low counts wereobtained from supernatants of cultures containingring forms and trophozoites. The highest counts wereobtained with the supernatants of cultures containingmature schizont segmenters and new ring forms afterreinvasion, at which time the TCA-precipitablepolypeptides in the supernatant represented 7% of the

106

P. FALCIPARUM POLYPEPTIDE RELEASE IN VITRO

Sequentiel incorporation and release of 35S-Methioninelabelled P falciparum polypeptides in synchronised culture

1 r-age of culiureihours) 0

parasitemia: 22. 9

ring torms'. 9 3. 7

t rophozoi tes: 6.3

schizonts

cpm incorporated in IRBC

cpm released in culture medium

-6 12 18 24 30 36 42

22.4 23.54 23.23 23.83

63.75 1 5.0 9.2 -

36.05 80 96.0 49.2- - 6.9 50.8

24.52 30.05

5.4 23.010.3 4

831 72.6

Table 1. Percentage of (35S)-methionine counts incor-porated in infected RBCs and released in culture super-natants

% counts' % counts'Labelling incorporated releasedintervals Developmental in infected in culture(hours) stages RBCs medium

0-6 Early rings 5.1 0.06

6-12 Rings 5.8 0.07

12-18 Rings + 11 5 0-07trophozoites

18 24 Rings+ 15.1 0.07trophozoites

24-30 Trophozoites + 11.4 0.1 5schizonts

30-36 Schizonts 10.2 0.36

36-42 Schizonts + 3.4 0.48rings

e 100% represents the total cpm added to each culture at thestart of the labelling.

Fig. 1. Incorporation of (35S)-methionine in infected RBCs(IRBC) and release of radiolabelled material in culturesupernatants during the asexual erythrocytic cycle. Eachof the successive individual cultures was incubated for 6hours with a constant amount of (35S)-methionine. At thebottom of the figure, the percentage of each erythrocyticstage of the parasite is indicated at various time pointsduring the experiment. Non-infected RBCs were alsoused as a control under identical conditions: there wasless than 0.5 x 104 cpm incorporated in normal RBCs andless than 0.2 x 104 incorporated in the supernatant.

radioactivity (as cpm) incorporated in the infectedRBCs (Fig. 1, Table 1).Both the parasitized RBCs (collected successively

during the asexual cycle) and the correspondingsupernatants were analysed by SDS-PAGE. Fig. 2Apresents autoradiographs of successive samples ofparasitized RBCs applied on SDS-PAGE. All thelabelled polypeptides were parasitized gene productssince mature RBCs are depleted of their genepool. Most of the P. falciparum polypeptides aresynthesized throughout the asexual cycle, as reportedpreviously (12). However, some polypeptides are syn-thesized or processed only, or mainly, at the time ofschizogony. Their localization on the gel is indicatedby arrows (Fig. 2A) and their relative molecularmasses (M,) are respectively > 200 000, 160 000,140 000, 105 000, 82 000, 55 000 and 41 000. Super-natants recovered from each of the successive cultureshave been analysed under identical conditions (Fig.2B). As would be expected from the results shown inTable 1, parasite-specific polypeptides are released insmall amounts in the supernatants before schizogony

and the number of detectable polypeptides increasesat the time of schizont rupture, which is when thesupernatant contains most of the schizont-specificpolypeptides. In addition, four polypeptides of Mr180 000, 76 000, 60 000 and 45 000 that are common

to the three developmental stages are clearlydetectable. Based on the intensity of the radiolabelledbands, polypeptides of Mr > 200 000, 180 000,140 000, 82 000, 60 000 and 45 000 were identified as

the major components released (Fig. 2B and Fig. 3,second band).To further identify the labelled polypeptides as

parasite-specific products and assess their immuno-genicity, the supernatants were used as a source ofmalaria antigens and immunoprecipitated by sera ofindividuals living in endemic areas, as shown in Fig. 3(bands Is, 2s, 3s). Lysates of unsynchronized,labelled infected cells were also used as a source ofantigens and immunoprecipitated by the same sera(Fig. 3, bands lc, 2c, 3c). The results demonstrate, onthe basis of the intensity of the polypeptide bandsdetected on the autoradiographs, that some malariaantigens are present in higher concentration in theculture supernatants than in the lysate of infectedRBCs. This difference is particularly evident for theschizont- and merozoite-specific polypeptides of Mr140 000, 82 000 and 41 000. Another schizont-specific polypeptide of Mr 160 000 is precipitatedsimilarly from both antigenic sources, whereas the Mr200 000 polypeptide is detected in higher concen-tration in the immunoprecipitates obtained by usingthe infected cells as a source of malaria antigens.

500-

x

100-

50-

10-

5-

10.7

L. RODRIGUEZ DA SILVA ET AL.

DISCUSSION

This study shows that, during the in vitro culture ofasexual erythrocytic stages of P.falciparum, parasite-specific polypeptides are released in the culturemedium. The results also confirm previous datademonstrating that the release of parasitic material isvery low during the maturation of ring forms andtrophozoites, increases at the end of schizontmaturation, and is maximum at the time of schizontrupture and reinvasion of RBCs by merozoites (4, 5).All the radiolabelled polypeptides are parasite-specific since mature human RBCs do not containnucleic acids. In addition, the radiolabelled poly-peptides released are precipitated by sera fromimmune individuals living in malaria endemic areas.In this study, the relative molecular masses of thereleased polypeptides were determined, two groupsbeing identified: polypeptides common to the variousdevelopmental stages of the parasites (with Mr180 000, 76 000 and 45 000, for example) andpolypeptides that are synthesized or processed duringthe schizont developmental stage. The polypeptideswith low Mr migrate with the front of the gels andprobably account for most of the TCA-precipitablematerial detected in the supernatants of culturescontaining ring forms and early trophozoites. Thismaterial probably consists of small polypeptidechains that leak from infected RBCs.The exact origin of the released polypeptides is

difficult to specify. They probably do not arise fromlysis of schizonts that fail to mature or from lysis ofmerozoites that fail to invade the erythrocytes, sincesome of the major polypeptides of schizonts are notrepresented in the supernatants, and since the cultureconditions have been optimized. One possibility isthat the released antigens represent residual parasiticmaterial that is not incorporated into schizonts.Alternatively, they may be parasite antigensassociated with the knobs present at the surface oferythrocytes containing schizonts, or antigensassociated with the coat of merozoites. The parasitepolypeptides common to the various developmentalstages probably represent parasite material that is notincorporated into the merozoites. The schizont-specific polypeptides have previously been identifiedand are shown in Fig. 2A (12, 13). They are goodcandidates for expression at the surface of schizontsand/or merozoites since polypeptides expressed late

in sporogony and schizogony have been shown inother plasmodia species to be expressed at the surfaceof sporozoites and merozoites and to be involved inprotective immune responses (14, 15). The apparentM, of the more intensively labelled schizont-specificpolypeptides are respectively about 200 000, 160 000,140 000, 105 000, 82 000 and 41 000. Some of thesepolypeptides are present in high concentration in thesupernatant of P.falciparum cultures at the time ofreinvasion, notably polypeptides of A(r 140 000,82 000 and, to a lesser extent, 41 000. Interestingly,these polypeptides have been shown to be the target ofmonoclonal antibodies that are able to inhibit the invitro growth of P.falciparum cultures (16). Thesemonoclonal antibodies are known to react withschizonts and merozoites, as demonstrated by theindirect immunofluorescence technique (13, 16). Ithas been shown by electron microscopy that mero-zoites release their coat during the invasion of RBCs(17). There is evidence here to suggest that thereleased polypeptides ofMr 140000, 82000 and 41000are part of the merozoite coat and may be compo-nents of the merozoites. In addition, the Mr 82000polypeptides may be identical with the knob-associated protein described by Kilejian (18). This ison the basis of its relative molecular mass and stagespecificity. It has not been elucidated whether or notthese polypeptides are intact polypeptides or frag-ments of a polypeptide of higher Mr, as shown inother malaria species (15). Comparison of the poly-peptide bands precipitated, using either infected RBClysate or culture supernatants, indicates that there isno (or only limited) additional degradation of malariaantigens in the culture supernatants, with the possibleexception of the polypeptide with Mr > 200 000.The importance of soluble antigens released by

haemosporidia during in vitro culture has beenemphasized by the recent demonstration thatvaccination with soluble B. bovis antigens derivedfrom cultures can induce protective immunity incattle (19). These experiments and other vaccinationexperiments, using soluble antigens of variousplasmodia (20, 21), suggest that the supernatants ofP.falciparum cultures may be a good source ofmalaria antigens that are suitable for inducing protec-tion. Soluble parasite antigens may also be of impor-tance for immunodiagnosis, since they may be used astarget antigens in serodiagnostic assays that aredesigned to detect current or recent malaria infection.

ACKNOWLEDGEMENTS

This work was financed by the World Health Organization, the UNDP/World Bank/WHO Special Programme forTropical Diseases, and the Swiss National Foundation (grant No. 3.890-0.82). The authors thank Ms E. Ramirez for herexpert technical help and Ms G. Fiaux for her excellent secretarial services.

108

APellet of cultures

240

67

43

30-

6 12 18 24 30 3.6 42

age of cultures(hours)

Fig. 2A. Autoradiographs (by SDS-PAGE analysis) of in-fected RBCs collected from successive culturesof P. falciparum, metabolically labelled with (35S)-methionine at 6-hour intervals. The arrows point tothe schizont-specific polypeptides with Mr> 200 000,160 000, 140 000, 105 000, 82 000, 55 000 and41 000.

-O.

0

E

B

240-

Culture supernatant

94-

67-

43 -

30-

6 12 I8 24 30 36 42 Pf

age of cultures(hours)

Fig. 2B. Autoradiographs (by SDS-PAGE analysis) ofsuccessive culture supernatants of P. falciparum, meta-bolically labelled with (35S)-methionine. The culturesupernatant corresponds to the infected cells analysed inFig. 2A. Identical volume of culture supernatant (8 Al)was applied on each lane of the gel. Pf: unsynchronizedP. falciparum-infected RBC preparation labelled with(35S)-methionine. The arrows point to polypeptides of M,> 200 000, 140 000, 82 000 and 45 000.

C,)

30

0E

220 -

o 194 -

67-

E43 -

36 -

C S ic is 20 25 30 35SNHS

Fig. 3. Autoradiographs (by SOS-PAGE analysis) ofimmunoprecipitates obtained by using immune sera andeither P. falciparum-infected RBCs (bands ic, 2c, 3c) orculture supernatants (bands is, 2s, 3s). Abbreviations:C = P. falciparum-infected RBCs; S = culture super-natant; NHS = normal human serum.

P. FALCIPARUM POLYPEPTIDE RELEASE IN VITRO 111

RESUME

CARACTERISATION DES POLYPEPTIDES LIBERESPAR PLASMODIUM FALCIPARUM LORS DE LA CULTURE IN VITRO

Des antigenes paludiques ont e mis en evidence dans leserum de malades souffrant de paludisme a P.falciparum.Certains de ces antigenes ont et retrouves dans le milieuemploye pour les cultures continues in vitro des formeserythrocytaires asexuees de P.falciparum.Pour les experiences presentees ici, des cultures syn-

chrones successives de P.falciparum ont et marqueespendant 6 heures A la (35S)-methionine. On a constate quel'incorporation de cet acide amine etait maximale a la fin dustade trophozoite. L'etude des surnageants des cultures amontre que tres peu de polypeptides marques etaient liberesdurant la maturation des formes annulaires et des tropho-zoites. Assez marquee A la fin de la maturation des schi-zontes, cette liberation devient importante au moment de larupture des schizontes et de l'invasion de nouveaux globulesrouges. On deduit l'origine parasitaire des polypeptidesmarques trouves dans le milieu de culture du fait qu'il n'y apas d'incorporation de (35S)-methionine en presence deglobules rouges normaux non parasites et que les polypep-tides marques liberes dans le milieu de culture sont precipitesselectivement par des serums humains d'individus partielle-ment immuns.

Les surnageants de culture et les globules rouges parasitesont et analyses par SDS-PAGE, suivi d'autoradiographie.Il apparait que les polypeptides liberes appartiennent A deux

groupes principaux: le premier comprend des polypeptidesqui sont synthetises et exprimes a tous les stades de deve-loppement &rythrocytaire asexue du parasite; le secondcomprend des polypeptides synthetises ou exprimes A la finde la schizogonie; ces derniers sont donc schizonto- oumerozoito-specifiques. On peut envisager que les polypep-tides liberes communs aux divers stades de developpementerythrocytaire (principalement polypeptides de Mr egale A180 000, 76 000, 60 000, et 45 000) representent du materielparasitaire non incorpore dans les merozoites; ce materielserait libere par l'eclatement des schizontes. Les polypep-tides schizonto/merozoito-specifiques dont la massemolaire relative (Mr) est de 140 000 ou 82 000 et, dans unemoindre mesure, ceux dont la Mr est de 41 000, sont particu-lierement abondants dans le surnageant des cultures. II estinteressant de relever que ces polypeptides sont reconnus pardes anticorps monoclonaux qui ont la capacite d'inhiberla croissance de P.falciparum in vitro. Ces anticorpsreagissent avec les merozoites en immunofluorescenceindirecte. I1 apparait donc que le surnageant des cultures deP.falciparum peut representer une source de materielinteressante pour la purification d'antigenes paludiquessusceptibles de jouer un role dans la reponse immune del'h6te.

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