Recent developments in production and purification of malaria antigens: Harvest of Plasmodium falciparum merozoites from continuous culture*

BzAllein of the World Health Organ,zauron, 57 (Suppl. 1) 63-6B (1979) Harvest of Plasmodium falciparum merozoites from continuous culture* J. E. K. MREMA,' G. H. CAMPBELL, ' A. L. JARAMILLO,2 R. MIRANDA,2 & K. H. RJECKMANN3 Spontaneously released merozoites were harvestedfrom cultures in which 42-90% of the erythrocytes had been infected with matureforms ofPlasmodium falciparum at the start ofincubation. The matureforms had been extractedfrom asynchronous cultures by the use ofFicoll and Plasmagel gradients. As the matureforms consisted ofboth rrophozoites and schizonts, merozoites were released into the culture medium over a longperiod of time. The synchrony ofmerozoite release didnot appear to be improved byprior exposure ofparasites to sorbitol. Over this prolonged period of incubation, the yield of merozoires was disap- pointingly low in cultures containing 2.5% of erythrocytes. Aft erythrocyte concentrations of0.01-0.250%, 3-10 times more merozoites were released into the medium; 0.4-2.3 mero- zoitesper initialmatureform were harvested over a 15-19-hourperiod. In addition to mero- zoites, contents of the culture medium included intact erythrocytes, ghost cells, and other cellularfragments. Only intact erythrocytes were effectively removedfrom the medium by simple or Ficoll gradient centrifugation. Merozoite preparations that are free from host cellular material are important in the development of a human malaria vaccine. in preliminary studies with blood-stage antigens of Plasmodiumfalciparuin, partial immunity to malaria was induced in Aotus monkeys after exposure to intra- cellular (1) or extracellular (2) merozoites. These anti- genic preparations contained various contaminants, including a considerable quantity of erythrocyte membranes. Such contaminants will probably have to be excluded from antigenic preparations that might eventually be used for human vaccination. Because of the shortage ofAotus monkeys, immunization studies with this scarce experimental model for falciparum malaria should, preferably, also be carried out with "clean" parasite preparations. Although partial pro- tection against falciparum malaria can be achieved by the use of saponin-lysed intracellular merozoites (1), it seems more desirable to pursue further studies with preparations containing spontaneously released merozoites (2) rather than to attempt the freeing and separation of intracellular parasites from their host cells. With the development of techniques for the con- centration of mature erythrocytic forms of P.falci- 0 From the Division of Tropical and Geographic Medicine, University of New Mexico School of Medicine, Albuquerque. NM 87131, USA. This work was supported by the Agency for Inter- national Development, US Department of State, Contract No. AID/ta-C-1259. Research Scientist. ' Biologist. Division Head. parum (3, 4) from continuous culture (5, 6), an increased number ofmerozoites can be collected from the medium because reinvasion is limited by the paucity of uninfected erythrocytes. Altbough a certain degree of separation of differ- ent parasite stages has been achieved (3, 4), cultures initiated with these "purified" parasite stages do not appear to be sufficiently synchronous to allow the harvest of a large number of merozoites over a short period of time. Limited investigations were therefore carried out to determine whether cultures initiated with ring forms might improve the synchrony of para- site development; ring forms were extracted from cul- tures after Ficol] centrifugation (3) or sorbitol treat- ment (Vanderberg, personal communication). Studies were also initiated to develop techniques for harvest- ing merozoites, relatively free of contaminating host cell material, for the preparation of malaria antigens. MATERIALS AND METHODS Source ofparasites Parasites of several strains of P.falciparum-FVO (Vietnam), FUP (Uganda), and FTA (Thailand)- were used in this study. They vere obtained from cul- tures that had been maintained in our laboratory by the candle-jar method (5). Parasites were cultured in 3869 - 63- 64 J E. K. MNREMNA ET AL. 60-mm Petri dishes containing a 2.5% or 5% suspen- sion of human erythrocytes in RPM1 1640 medium. Each ml of medium included 0.1 ml of human serum, 5.94 mg of HEPES buffer, 2 mg of sodium bicar- bonate, and 33 ,g ofgentamicin sulfate. When mature forms (MFs), either trophozoites or schizonts, reached a concentration of2-5%,they were extracted by Ficoll" gradient centrifugation (3) or by a 3%o solu- tion of gelatin in physiological saline-.Plasmagelb (4; W. Trager, unpublished observations, 1978). Ring forms (and some young trophozoites) recovered in the bottom fraction of the Ficoll gradient columns were so - , o4C -- 9 T TT 10 4i°C / *RING FORMSD-'-O MATURE FOAMS 260 20 24 48 12 96 12 144 1 TIME OF INCUBATiON (HOURS) Fig. 1. Growth of P. falciparum (FVO strain) in continuous culture afterstorage of the culture {in a candle jar) at4 oC for 2 days. Before storage, parasites were grown at 37 °C for 48 h until most parasites were ring forms. At the end of cold storage, the cultured parasites were reincubated as usual. Each point on the graph represents the mean of 3 replicate cultures in 60-mm Petri dishes. ° From Sigma Chemical Co., St Louis, MO, USA. b From Laboratoire Roger Bellon, NeuiLly, France. subcultured and reincubated for at least 1i develop- mental cycles before further MFs were extracted from the subsequent harvest of merozoites. Altematively, parasites recovered from the bottom fraction of the gradient were stored temporarily at 4°C for 2 or 3 days (Fig. I) and MFs were extracted by gradient cen- trifugation after further incubation for a few days. Exposure ofparasites to sorbilol About 9 ml of an erythrocyte suspension from a primary culture were centrifuged at 350 g for 10 min. After removing the supematant, an equal volume (9 ml) of 50 g/litre (5%) aqueous sorbitolc solution was added to the pellet, the sediment was resus- pended, and the suspension was allowed to stand for about 5 mn. The preparation was then washed twice with RPMI 1640 medium and incubation of the para- sites was resumed. The in vitro growth of these sorbi- tol-exposed parasites was then compared with that of parasites recovered from the bottom fraction of the Ficoll gradient (3). Harvest ofmerozoites Short-term secondary cultures were initiated with Ficoll- or Plasmagel-concentrated preparations of in- fected erythrocytes containing 42-90% MFs. These erythrocyte preparations were then resuspended in culture medium at a concentration of 0.01-2.5% and incubated under the same conditions as primary cul- tures.-The development of parasites and the release of merozoites were monitored by examination of parallel microcultures (7) at various times during incubation. At appropriate times after the onset of incubation, culture specimens were centrifuged at 150 g for 10 min and free merozoites were recovered from the supernatant medium. Erythrocyte pellets were then resuspended in fresh culture medium and incubated to allow more parasites to mature to merozoites. Removal of erythrocytes Separation of merozoites from erythrocyte com- ponents was attempted by consecutive centrifugation of supernatant media at 200 g and 250 g for 10 min. Supernatants were then examined for merozoites, in- tact erythrocytes, and ghost cells. Separation of merozoites from erythrocytic material was also at- tempted by layering I ml of a merozoite suspension (containing at least 1 x 107 merozoites) on top of4 ml of a 5% Ficoll gradient in a conical centrifuge tube. After centrifugation at 600 g for 10 min, the gradient was aspirated (exclusive of the pellet) and centrifuged at 3000 g for 10 min. The pellet was then resuspended in 1 ml ofmedium and examined for cellular elements. ' Fromn Sigma Chemical Co , St Louis, MO, USA. HARVESTING P. FALCIPARUM MEROZOITES FROM CULTURE Enumeration ofmerozoiles and erythrocytes The concentration of merozoites, intact erythro- cytes, and ghost cells was determined by examination of wet fldms in a thin Neubauer haemocytometer. The percentage of infected erythrocytes, containing dif- ferent developmental asexual stages of the parasite, was assessed by examination of 500 erythrocytes in Giemsa-stained thin blood smears. RESULTS Growth ofparasites after exposure to sorbitol As first observed by Vanderberg (personal com- munication), schizonts and mature trophozoites were destroyed after exposure of primary cultures to sorbi- tol. The remaining ring forms, of various ages, and young trophozoites were subcultured and the syn- chrony of parasite development was comparable to that observed in cultures that were started with ring forms obtained from a Ficoll gradient (Fig. 2). By 43 h 0-- uz I 15 Li 10 a C] z. a * RING FORMS TROPHOZOITES - * SCHIZONTS AB AB AE 0 19 43 t I I I A B 72 TIME OF INCUBATION (HOURS) Fig. 2. Growth in vitro of P. falciparum nng forms after primary cultures containing asynchronous parasites were either treated with sorbitol (A) or extracted with Ficoll (B). The development of parasites was assessed by examination of 3 replicate cultures. after the start ofculture, the vast majority of parasites were ring forms at different stages of development. As maturation from young to old rings may extend up to a 24-h period of the asexual erythrocytic cycle, in- fected erythrocytes, at 72 h, contained schizonts, trophozoites, or newly invaded ring forms. Parasite development in these cultures was not sufficiently synchronous to enable the collection of a large number of recently released inerozoites over a short period of time. -. sw. Fig. 3. Ficoll-concentrated mature forms of P. falciparum obtained from an asynchronous culture in which 3.2% of erythrocytes contained mature-form parasites. Harvest of merozoites Fig. 3 depicts MF parasites that were concentrated by Ficoll or Plasmagel. The proportion of infected erythrocytes varied from 42% to 90% and, in most of the experiments, more trophozoites than schizonts were present in the final preparation. During the incubation of these parasites, the culture medium as- sumed a dark-brown colour owing to the formation of pigment by maturing parasites. Examination of blood smears at various times during incubation showed maturation of trophozoites to schizonts and an increasing proportion of mature schizonts. Table I shows the number of merozoites released into the culture medium after incubation of Ficoll- concentrated MFs for various periods of time. The re- sults indicate that the yield of merozoites was low, but 65 66 J.E.K RENMAETAL Table 1. NMerozoites harvested from cultures started with Ficoll-concentrated mature forms of P. falciparum Mature forms at No of merozoites (cumnulailva)Cortcantratson atari of culture harvstd per initial mature forn Expenment Strain of of erythiocytes _ _ _ _ _ _ _ No P falcpwun in medium ( x 10-'/ml Schizonts 1%) after 4-8 hoursa after 19-27 hourss 1 FVO .2.50 168 12 0 01 0.16 2 FV0 2.50 154 22 0.05 0.20 3 FVO 2 50 150 10 0.09 0.10 4 FVO 2 50 175 19 - 0.16 1.25 88 19 - 033 5 FUP 2 50 167 6 - 0 08 1 25 79 6 - 017 062 39 6 - 048 a Tne from start of culture Table 2. Morozoites harvested from cultures started with Plasmagel-concentrated mature forms of P. falciparum Mature forms at No of merozoites lcurnulativeConcentration start of culture harvested per initial mature form Experimnent Strain of of erythrocyte __ _ _ NJo. P falpkom nmedium t%) x 10 4/ml Schzonts t%l after6-8 hoursa after 15-19 hours5 1 FUP 2 20 114 27 0 11 0.15 0 20 19 27 0 74 1.32 2 FUP 0 20 B 15 055 071 0.02 08 15 075 087 3 FUP 0 20 11 28 039 0 83 4 FUP 020 14 38 - 0.75 5 FUP 0 20 11 19 - 0.40 0 02 1 19 - 1.00 6 FVO 2.50 224 23 0.06 0 10 1.2S 112 23 0.09 013 0.25 22 23 0.21 0.44 002 2 23 050 1 10 7 FVO 0 20 1 8 29 0 42 0.64 0 10 9 29 0 44 0.64 001 s9 29 067 0.77 8 FTA 250 153 15 - 0.11 0 20 12 15 - 1.28 0.01 0.6 15 - 2 33 a Timoe from start of culture that more merozoites could be harvested from cul- mature form within 15-19 h after the start of incu- tures containing fewer erythrocytes. Table 2 shows bation. This yield of merozoites was 3-10 times the number of merozoites collected from Plasmagel- higher than that observed from cultures containing concentrated MFs after incubation at lower erythro- 2.5% of erythrocytes. In these cultures started with cyte concentrations. At the higher concentrations, the erythrocytes containiing 42-90%to of MFs, numerous yield of merozoites was comparable to that obtained degenerating parasites were observed during the from Ficoll-concentrated MFs (cf. Table 1). The yield course of incubation at higher erythrocyte (or para- of merozoites was increased by lowering the concen- site) concentrations. This probably accounted for the tration of erythrocytes; at concentrations of 0.01- few free merozoites and the low reinvasion of un- 0.2511, 0.4-2.3 merozoites were harvested per initial infected erythrocytes observed in such cultures. In HARVESTING P. FALCIPARUM MEROZOLTES FROM CULTURE Table 3. Attempts to separate intact erythrocytes and ghost cells from P. fa(ciparum merozoites by simple centrifugation for 10 minutes Concentration {x 10/1/ml) Recovery after centrifugation Expenrment Force of centnfuation Source of before cenirifugaton % No (g9 matenal Mero- Ghoet Intact Mero- Ghost Intact zuites cells erythrocyles zoites cells erythrocyes 1 150 Whole culture 14.7 198a 7 73 58 <1 200 Supematant 10.3 115 <1 56 50 0 2 150 Whole culture 8 0 17 0. 40 8 94 55 1 200 Supernatant 7.6 9 3 <1 93 38 0 3 150 Supernatant 12.3 14 8 <1 73 80 <1 200 Supernatant 9.0 118 <1 49 52 0 250 Supernatant 4.0 4 7 <1 55 47 0 4 150 Supematant 113 11 6 <1 97 83 <1 200 Supematant 11.1 95 <1 80 78 0 250 Supematant 8.8 7 4 <1 94 89 0 5 250 Supematani 4 3 4 7 <1 52 56 <1 7 150 Whole culture 56 10 5 7 3 93 31 <1 8 150 Whole culture 25 8.1 110 88 50 <1 Table 4. Attempts to separate intact erythrocytes and ghost cells from P. falciparum merozoites by Ficoll gradient centri- fugation for 10 minutes Concentration Ix 10-Imlm Recovery after centnfugation Experiment Force of centrifugation Source of before centniugation M% No lgl matenal Mero- Ghost Intact Mero- Ghost Intact suites cells erythrocyle3 zones cells erythrocyles 1 600 Whole culture 1.7 0.1 28 72 50 0 2 600 Supernatant 2 1 4.3 <1 91 66 0 3 600 Whole culture 3 5 2 1 15s 98 62 <1 4 600 Supematent 4 0 26 0.4 95 46 0 some of these cultures, spberical structures (piobably fragments of infected erythrocytes) were observed oc- casionally. Although they could be readily differen- tiated from merozoites in stained smears, special care was taken not to mistake them for merozoites during examination of wet films. Removal of erythrocytes Table 3 shows the results obtained after centrifu- gation of whole cultures, or supernatants from cul- tures, at 150 g to 250 g for 10 min. After centnfu- gation at 150 g, 990% or more of the initial erythro- cytes and 17-69%o of the ghost cells were removed from the specimens. Between 3 and 27°7o of the mero- zoices were also lost during this procedure. Although very few intact erythrocytes remained in this mero- zoite preparation a substantial number of ghost cells were still present. Further centrifugation of these pre- parations at 200 g and 250 g usually resulted in a de- crease in both ghost cells and merozoites. Hence, there was no advantage in applying a centrifugal force in excess of 150 g. Table 4 depicts the results of merozoite specimens obtained after centrifugation of a whole culture of supernatant in a SGo Ficoll gradient. Most of the merozoites were recovered in the top fraction of the gradient column. The merozoite preparations were, however, contaminated with ghost cells and other cellular fragments. The denser intact erythrocytes, some pigment, and 34-540/o of the ghost cells were collected in the pellet at the bottom of the tube. These findings indicate the need for further investigations to eliminate all ghost cells and other cellular elements from merozoite preparations. 67 68 J.E. K. MREMAETAL. DISCUSSION These studies demonstrate that free merozoites can be collected from cultures that were started with Ficoll- or Plasmagel-concentrated mature forms of P.faciparum. Although the synchrony of parasite maturation and reinvasion is improved by the use of sorbitol or Ficoll, cultures are still not sufficiently synchronous to permit the collection of a large number of merozoites over a short period of time. In our studies, merozoites were released slowly over a prolonged period and the total yield of merozoites was disappointingly low. Although the latter phenomenon may be partly explained by a degener- ation of some merozoites in the interval between examinations, e.g., between 4 and 27 Ih after the onset of culture, it seemed that the low merozoite yields were probably related to other factors. The degeneration of mature forms, particularly of actively growing schizonts, suggested that cultures contained too many infected erythrocytes. By lowering the number of erythrocytes in culture from 2.5% to 0.25% or lower, much higher yields of merozoites were obtained. By constant replenishment with fresh culture medium, it may be possible to raise the con- centration of MFs without depressing the maturation of schizonts and the subsequent release ofmerozoites. Merozoites were retrieved from culture in the supematant after centrifugation at 150 g for 10 min or in a Ficoll gradient column after centrifugation at 600 g for 10 min. Although more than 99% of intact erythrocytes were removed by these procedures, a substantial number of ghost cells, cellular debris, and pigment could not be excluded from the merozoite preparations. Further centrifugation of supernatants at higher g-forces was of no practical value in achiev- ing further separation of merozoites from erythro- cytic components. Additional investigations to obtain merozoite preparations that are free from host cellu- lar material are important for the ultimate develop- ment of a human malaria vaccine. RESUME RECOLTE DE MP-ROZOITES DEPLASMODIUMFALCIPARUMEN CULTURE CONTINUE Des merozontes libris spontan6ment ont ft6 rcolt&s dans des cultures faites A partir d'erythrocytes dont 42-9O074 itaient infectEs par des formes adultes de Plasmodiumfaci- parum au debut de l'incubation. Ces stades adultes du para- site avaient ete extraits de cultures asynchrones en utilisant des gradients Ficoli et Plasmagel. Comme les formes adultes comprennent aussi bien des trophozoites que des schizontes, la libkration de m6rozoltes dans le milieu de culture s'Etend sur unelongue pbriode de ternps, ct la synchronie dans la libb- ration des m&rozoJtes n'a pas paru ctre am6lioree par une exposition pr6alable des parasites au sorbitol. Avec un taux d'6rythrocytes de 2,5'o dans les cultures, la production de mtrozoites ainsi 6talEe sur une longuep6node d'incubation a WtE plus faible qu'on ne ]'escomptait. PoLur une concentration d'erythrocytes deO,01-0,2507o, 3 i 10 fois plus dc m6rozoltes ont W lib6bs dans le milieu; la proportion de merozoltes r6- coltEs au cours d'une p6riode de 15 A 19 heures pour chaque forme adulte initiale a W de 0,4-2,3. Outre des merozoites, le milieu de culture contenait des erythrocytes intacts, des cellules fantomes et d'autres d6bris cellulaires. Seuls les ery- throcytes intacts ont pu etre extraits efficacement du milieu au moyen d'une centrifugation simple ou faite dans un gradient Ficoll. Pour la mise au point d'un vaccin antipalu- dique humain, il est essentiel de disposer de pr6parationsde m6rozoites d6barrass6es de tout fragment de mati&e pro- venant de la cellule hote. REFERENCES 1. SIDDIQUI, W. A. Science, 197: 388-389 (1977). 2. MITCHELL, G. H. Er AL. Lancet, 1: 1335-1338 (1977). 3. MREMA, J. E. K. ET AL Bulletin of the World Health Organization, 57:133-138 (1979). 4. PASVOL, G. ET AL. Annals of tropical medicine and parrasitology, 72: 87-88 (1978). 5. TRAGER, W. & JENSEN, 1. B. Science, 193: 673-675 (1976). 6. HAYNES, J. D. Er AL. Nature (London), 263: 767-769 (1976). 7. RIECKMANN, K. H. ET AL Lancet, 1: 22-23 (1978).