Recent developments in production and purification of malaria antigens: Isolation of stages of the human parasite Plasmodium falciparum from culture and from animal blood*

Butellet of the World)Ieatlh Org,anozow, 57 (SuppI [)- 53-61 (1979) Isolation of stages of the human parasite Plasmodium falciparum from culture and from animal blood* ROBERT T. REESE,' SUSAN G. LANGRETH,' & WILLIAM TRAGER2 Procedur-es for isolation of various forms of the asexual erythrocytic stages of the human parasite Plasmodium falciparum are outlined. The procedures etnploy the plasmna expander Physiogel, which is composed of a chenmiclly modified, partially hydrolysed gelatin dissolved in Ringer's lacrate. Based on the observation that parasitized cells which are easily separable by this techzniqLte differ appreciably at the ultrastructural level, a mnech- anism by which separation occurs is proposed. Until recently, one of the major obstacles impeding research on malaria lhas been availability of parasite mnaterial. This problem had forced workers either to conduct studies in endemic areas or to maintain the parasite in thc laboiatory by continuous animal pass- age (1). The development of in vitro techniques tor cultivation of malaria has donc much to eliminate this problem (2). CulturC systems can be used as a means of both producing specific stages of the parasite and studying the organism undei controlled conditions. Nevertheless, at prcsent there are still limitations. Strains of Plasmnodiumn falciparum that have been grown in culture for more than a few cycles gencially do not develop in a completely synchronous fashion Analysis of any alhquot from a culture is likely to ieveal nmembers of all of the asexual erythrocytic stages of the organism. Foi strains that have been in culture for extended pceiods the sexual, gametocyte, Iorm is seen only rarely. Thuis, procedures must be dc- veloped to alloxv isolation ol specific stages of Lhe parasite To bc most useful, the piocedures should not adversely affect parasite 'iablity. For some time it has been knov n that red cell aggre- gation could be induced by mixitig with molccules that could bind wveakly to the red cell burface (3) Coulson & Chalmers took advantage ol this method ol selec- tive sedimentation of red blood cells to isolate lyni- phocytcs (4). More recently. Pasvol et al. (s) ha%e * From ilie Laboratory of Paiasitolou:, The Rockefcller Uni%er- sily, 1230 York Avenuc, New Y'ork. N; 10021, U.SX The iavevti- gdiion wasulpporicd bi Graint T16,181 'MV2 17tB) frond -lie UNDP/ Wotld Bank/WHO Special Programme ror Research anid Training n Tropical Diseases, Grant Al -08989 tromrNiAID. US Pti6lic HJallii Seivice, an(l Contract Ta-C-1373 frontt the US \genc% toi lrter- national Dcvelopmenit Assistant Professor ' Professor and Hcad of Laboratorv used a plasma expander, Plasmagel," ro concentrate red cells infected wiLh P.falciparum whiich had been obtained from human blood. Plasmagel is a 30 M/litrc .,olution of gclatin in physiological saliic. It is often difficult to obtain, thus a study was begun lo test a simldar product markctcd under the name, Physiogel SRK.0 The use of this material to isolate various stages of the patasite vill be described. MATERIALS AND MIETHODS Soturce ofparasitized red blood cells The organism used for most of these studies was the FCR-3/FMG strain of P.jalciparumn. Two other strains (FCR-l/FVO and FCR-4/6252) lhave givcn similat results. Parasites were grown in viitro in lhuman A Rh + cells (2) or wcre obtained froin the blood of infected Aorus monkeys. Getatini conmpound usedfor separalions The gelatin plasma expander Physiogel SRK was used for all cell separaLions. The gelatin used in this plasma expander had been chemically modified and partially hydrolysed to give moleculcs with an avei age relative molecular mass of approximately 22 000. Physiogel is a 401 solution of this low-molecular- weight gelatin in Ringer's lactate. It is a relatively inexpensive rnixture having more versatile physical properties than typical gelatin solutions. Because of the physical and chemical modiftcations of the gelatin it can be used even at 4 C without fear of gelation. F.:oni L31)oratoire RoLcr Bellon, Neudilv. FranLc. F.o:rw Zntrai1l.kbo ao:-iun, IMluispendcdicnsr SRK, 3000 Berne s22 S:-:tzclaiid 3868 -53 - R. T. REESE ET AL. Counting ofparasites Parasitaemias were determined on Giemsa-stained thin smears. The number of parasitized red blood cells observed in a minimum of 50 fields was deter- mined relative to the total number of red blood cells present. Electron microscopy Fresh parasite material was prepared for electron microscopy as previously described (6). Sections were examined in a Philips 300 electron microscope. RESULTS Physiogel separation procedure Mixing of gelatin compounds such as Physiogel witb parasitized blood enhances the sedimentation of those cells that retain the essential physical properties of a normal red cell. Those cells containing mature parasites remain in the upper phase. Sedimentation is rapid enough at 1 g that centrifugation is not required. Cells remaining in the upper phase (most of which will contain mature parasites) can then be collected by low-speed centrifugation (550 g). To determine the conditions under which separ- ation is optimal, the first experiments were conducted using 25% cell suspensions and allowing settling to occur for 30 min at 37 'C. These experiments demon- strated that a ratio of approximately 50%o Physiogel: 50% cell suspension was best. The remaining experi- ments were therefore conducted using mixtures in which the final Physiogel concentration was 50%o. To determine the effects of time and temperature on the selective isolation of red cells containing mature parasites, parasitized red cells obtained from culture (4% parasitaemxa) were suspended in medium without serum to give a 25% cell suspension. Aliquots (1.5 ml) were dispensed into test-tubes followed by equal volumes of Physiogel. After allowing the con- tents of each tube to achieve the desired temperature (4 'C, 26 'C, or 37 °C), the Physiogel-cell suspensions were mixed and left for the specified times (15 , 30, or 45 min). The unsedimented cells in the upper phase were removed by Pasteur pipette, collected by centri- fugation, and smears were made. The percentages of parasitized cells that remained in the upper layer at each time and temperature are shown in Table 1. Thc data demonstrate that the rate of settling of normal red blood cells is increased by increasing the tempera- ture. Optimal separation appears to occur in approxi- mately 30 min at 37 'C or 45 min at room temperature (26 'C). The following standard conditions have been adopted: Physiogel mixed with a parasitized red cell suspension to give a final concentration of 12.5%o cells at50% Physiogel. Settling is allowed to occur for 30 mi at 37°C. Table 1. The effects of time and temperature on the propor- tion 1%) of parasitized red cells remaining among the un- sedimented calls in the upper phase Time Temperature (mri'nl4 OC 26 DC 37 OC 15 8 32 46 30 30 46 61 45 41 62 57 It should be noted that maximum separation using such small aliquots of cells often results in Final sus- pensions that contain approximately 60% parasitized red cells. When experiments are conducted witb larger volumes of cell suspension containing about the same initial percentage of parasitized red cells, the final parasitaemia of the unsettled cells is generally higher (80-90Oo). That efficient separation appears to occur best when relatively large volumes of parasitized cells are used, makes this a particularly useful technique for preparative isolation of cells containing mature forms of the parasite (trophozoites, schizonts, and segmenters). Although Physiogel separations can be used as a means of concentrating parasitized red cells, the pro- cedure clearly does this in a selective fashion. Those parasites which by light microscopy are still at the ring stage settle with the normal red cells (Fig. 1). Para- sitized red cells containing more mature parasites remain in the supernatant (Fig. 2). Electron micro- scopic examination of parasitized cells from the top and bottom phases after mixing with Physiogel pro- vided evidence of obvious physical differences in the cells separated. As seen in Fig. 3, those parasitized red cells that did not settle had surface modifications which have often been characterized as "knob-like". In contrast, those parasitized cells that did settle along with the normal red cells, had no obvious surface modifications. These data demonstrate two things: that parasite- induced alterations of the host red-cell membrane coincide with the light microscopic event of changing from a ring form to a trophozoite; and that the red cell containing the young parasite that is by light microscopy a ring form, is sufficiently different physically from that containing the more mature forms of the parasite (trophozoites, schizonts, and segmenters) that sharp separation of these forms can be effected by simple sedimentation in gelatin com- pounds such as Physiogel. 54 *In fl ca, ca CD (D() c)0 0 o 0 3 3 0 S 5 (D CD 0 coOC (D CD -D CD :r =r CO 0 0 -< < ~. 0 o a- 0 0 h10 0o tD oS 0 O 0 o 0 M 0 0 e. C O CLD C5,N N CA)0 o CD -z - CO co 0 0. 0. ;L 5~ : 0 3 0 PF CD 0 0L- CC C,OD 00~ 0 W N;00 0 g0 0 * -4- 3 o ..~ 0 C0 ~- 0 '40.S 00=. I F 0 Fig. 4. Early schizont from Physiogel upper phase. The ultrastructure is unaltered by the separ- ation procedure. X 19 200. Fig. 5. Segmenter from Physiogel upper phase. The fine structure is normal. X 20 250. 0) ) ',i IOOo a:F E 2 - O. 0000 .n I~~~~ ~~~ --3co c.:r 02 o~u1 to _DC _ ;6 0 o O. o~j~Eo < a .ar Eo,a 0 ) E1);z +--. -'-- O *_ ob ° , U- @, 'a0 C~~~~~OMI.JCl3C-ZWCD 94 W ~~~ ~~ 0C 0 I ~~~~ o"; C *}~~~~~~~~~J. c u - - a,~~~~W. - o *, 0 E 0 0 * * (0 00- b wOo -~~~~. L- 0 CO 0m V*C: b F C~~~0(i CD °~os C ' E U .~~~. u E # .*Xt ,@C,.L a) D oE cn o CL.2 c O 0 0 C0 .E 0 CD O s M O E0 C _ = > Z '.c*, 0_ X .' SLXaxp.. coo ' a:, .,. U D .EVOm - rLaIC O, D .1 ;' a ,1 IFig. 10. Extracellular parasites prepared by the Physiogel-saponin method. This preparation contains schizonts, segmenters, and merozoites. There are some contaminating membranes, including erythrocyte membranes, and some unidentified debris. X 12 000. 4. N-.. :.., '.. ,` .. ':. r -;#.k. 4 ISOLATION OF P. FALCIPARUM STAGES FROM CULTURE OR BLOOD 59 Fig. 3-6 are electron micrographs of parasitized cells obtained by Physiogel separation. The parasites have not undergone any noticeable change due to the procedure (6). Confirming the benign nature of the separation procedure, when parasites isolated from either the top or bottom phases are returned to culture, their viability does not appear to be adversely affected. This is of considerable importance when working on a preparative scale for the isolation of antigen. Preparative Physiogel harvesting of parasitized red cells Physiogel can be particularly useful when relatively large volumes of parasitized red cells must be selec- tively isolated to obtain antigens for immunological studies. It should be equally useful to the biochemist interested in metabolic studies. In this way one can obtain a highly concentrated suspension of parasit- ized cells which are restricted in their stage heterogen- eity in a fashion likely to be reflected also by the presence or absence of specific metabolic pathways. The procedure may be used in two ways depending on whetber the parasite source is an in vitro culture or an animal. Harvestfrom in vitro cultures. Parasites are grown in culture until 10-20'o of the red cells are para- sitized. Upon reaching this point, the growth stage of the parasites is then monitored until the proportion of the cells containing segnmenters reaches a maximum. Physiogel harvesting of the red cells containing mature parasites is conducted by the standard pro- cedure allowing settling to occur in 40-50-mI volumes. Unsedimented cells in the upper phase are removed and then collected by centnfugation (550 g). Those cells that sediment rapidly are returned to cul- ture to be harvested the next day after appropriate maturation has occurred. The parasitized cells har- vested the second day are often more synchronous than those harvested initially. Harvest from infected aninal blood. When para- sites are to be harvested from the blood of infected animals, a somewhat different procedure is used. This is dictated by the nature of the starting material, which is likely to contain a relatively large number of white blood cells but very few mature forms of the parasite. Although white cells can be removed in a nunber of ways, use of Physiogel according to the standard procedure is an excellent one. After mixing of the blood with Physiogel, virtually all the parasitized red cells will settle because they will be at the ring stage. The white cells remain suspended, as do the few red cells that contain mature parasites. Thus, careful re- moval of the upper phase will eliminate the contami- nating white cells, leaving a fairly homogeneous group of parasites at the ring stage. If more mature parasites are needed, short-term cultures can be estab- lished to allow maturation to the desired stage. Maturation to the segmenter stage often requires more than 24 h. Harvest of merozoite-rich fractions When merozoites are desired for immunological or other purposes two general procedures have been used to obtain them. The first procedure is useful when mature merozoites are needed showing little or no ultrastructural deterioration. It is more time-con- suming and the yield of parasite material is often lower. The second procedure is useful for preparative isolation of parasite material. (a) To obtain merozoites showing little deterior- ation at the ultrastructural level, it is generally best to harvest by allowing normal maturation of the seg- menters. This can be accomplished by transferring the red cells containing relatively mature parasites that have been isolated from the Physiogel upper phase to a small Erlenmeyer flask with a cotton-wool stopper. Complete medium containing 100 ml of human serum per litre is added so that the cells constitute only 5- 101o of the total volume of the suspension. After gentle swirling to distribute the cells evenly through the suspension, the culture is placed in a candle jar and incubated at 37aC. If the merozoites released during a 2-h period are to be barvested, it is useful to resuspend the cells by swirling every 20-30 min. Be- cause there are only a few normal red cells available to bind merozoites and these are widely dispersed in the suspension, most of the merozoites released can later be collected by centrifugation. A sample of mero- zoites harvested in this way is shown in Fig. 7. (b) A second method of harvesting that is particu- larly useful when parasite material is required for im- munological work, employs a saponin detergent.c Again, cells from the Physiogel upper phase are used. They are suspended in an equal volume of a high- potassium buffer (7) that is used throughout this pro- cedure. The cell suspension is then mixed with 2 vol- umes of a 0.03 07o saponin solution (final saponin con- centration 0.015%) and left at 37 0C for 10 min. Eight volumes of a 5% bovine serum albumin (BSA)d- buffer solution are added, and the suspension is then passed under pressure through a 23-gauge syringe needle to rupture parasitized cells weakened by the detergent. Intact cells are removed by centrifugation at 440 g for 10 min, after which the released parasites are collected by centrifugation for 5 min at 4200 g. To c From EasLman Kodak, Rochester, New York, USA. d From Rekeis Chemical Co . Phoenm, Arizona, USA. R. T. REESE ET AL. reduce the number of contaminating red cell mern- branes the parasite material is washed twice in the 5%o BSA. A final wash in buffer alone reduces the remain- ing BSA appreciably (Fig. 8). Parasite material iso- lated in this way is composed of trophozoites, schizonts, segmenters, and free merozoites (Fig. 9). Membranes, some of which are from red cells, are also detectable when preparations are examined by electron microscopy (Fig. 10). Although quite crude, this material is a good source of parasite antigens and has been used successfully to immunize Aotus monkeys against P.falciparum even without the need for complete Freund's adjuvant (8). DISCUSSION One of the main factors that has impeded research on malaria has been the difficulty of obtaining para- site material for study. In 1967, Geiman & Meagher (1) found that Aotus monkeys could be infected with P.falciparnm and that this generated a disease similar to that observed in humans. This not only provided an animal model for falciparum malaria but estab- lished a means by which investigators working in non- endemic areas could have ready access to organisms for study. Because a number of limitations are im- posed by having to maintain any parasite in an animal, particularly a primate, more rapid progress was stimulated by the development of a method by whicb the organism could be continuously grown in vitro (2). Since that time, there has been a major influx of new scientists into malaria research, each with his own particular need for a specific stage or parasite-induced component. In vitro cultures of P.falciparum can provide a good source of the parasite-associated components that are synthesized during the asexual erythrocytic cycle of the organism. The problem faced by most in- vestigators is how to obtain exactly what they need from a culture in which the parasite does not grow in a synchronous fashion. Equally troublesome has been the isolation of parasite material from the blood of animals without considerable contamination with white cells and platelets. The present work has been concerned with a number of these problems. It has provided rapid and simple ways of separating either large or small volumes of erythrocytic forms without obvious harm to the parasite. Physiogel fractionation separates red cells containing parasites that by light microscopy are rings, from those containing trophozoites, schizonts, or segmenters (Fig. I and 2). Electron microscopic studies have demonstrated that the clear distinction between the light-microscopic forms is paralleled by a distinction between parasitized cells with normal and modified surfaces (Fig. 1-6). Thus, ring forms that have not altered the red cell surface are aggregated with normal red cells, while the more mature parasites that have altered the red cell surface are not. For some time it has been known that fibrous proteins such as fibrnogen and gelatin, as well as other high-molecular-weight molecules such as dex- trans, can bind to red cell membranes effecting cell aggregation (3). Molecules with high axial ratios are particularly useful because of their ability to cross- link cells and still retain a relatively large intercellular distance. This helps to reduce the repulsive forces between cells that are created mainly by the sialic acid residues covering the red cell surface. Thus, the mixing of gelatin, in the form of Physio- gel, with parasitized blood probably leads to binding of the protein to the surface of red cells followed by cross-linking. Those cells, whether normal or para- sitized, that retain sufficient defornability to stack as concave discs, aggegate forming rouleaux. As is pre- dictable, increasing the temperature increases the sedimentation rate in the presence of such molecules (Table 1). In the case of gelatins, there are two possible explanations: increasing the temperature serves to lower the viscosity of the gelatin solution, allowing more rapid interaction among cells; and, in addition, increasing the kinetic energy of the system directly increases the movement of both molecules and cells, thus increasing their interaction. Neverthe- less, because of the physical and chemical properties of the gelatin used in Physiogel, the influence of temnperature on such mixtures is considerably less than would be expected with mixtures of higher mol- ecular weight gelatins (3). With some understanding of how Physiogel in- creases the rate of red cell sedimentation, why then does it allow separation of cells containing parasites at different stages? An answer is provided by examining the results of the electron microscopic study. Parasitized red cells whose morphology has not yet been altered by parasite development (i.e., by the development of knobs) apparently retain enough of the physical properties of normal red cells to form rouleaux and sediment. In contrast, parasites that have developed past the ring stage generally induce major alterations in the morphology of their host red cell and also effect changes in its membrane (6). These, together with the constraints imposed on the flexibility (deformability) of the red cell as the para- site constitutes an ever increasing proportion of its interior, must sufficiently reduce its ability to stack properly that it is excluded from the rouleaux, thus re- mainng in suspension. This difference in the physical properties of these two groups of cells (ring forms and mature parasites) provides a powerful tool for obtain- ing a fairly homogeneous sample of erythrocytic forms of P.falciparum. 60 ISOLATION OF P. FALCIPARUMf STAGES FROM CULTURE OR BLOOD 61 ACKNOWLEDGEMENTS We wish to thank Dr Woods for supplying the PhysLogel used in this study. We also thank Mrs Greene and Mr Borrero for their help with the work and with preparing the manuscnpt. RESUMvE ISOLEMENT DE DIVERS STADES DU PARASITE HUMAIN PL4SMODIUMFALCJPARUM A PARTIR DE CULTURES OU DE SANG D'ANIMAL L'examen au microscope 6lectronique de globules rouges infect6s soit par de jeunes parasites Plasinodiwm falci- parum, soit par des parasites adultes a moontr6 qu'il existait, entre les deux types de cellules, des difr6rences physiques appr6ciables, et cette constatatLon a permis de mettre en euvre le mecanisme de 46paration en deux groupes des erythrocytes auquel font appel les mnthodes d6crites dans la pr6sente etude. On a utilis6 Physiogel, solution A 4% de g6latine cbimiquement modifi6e, partiellement hydroJysEe et dissoute dans le lactate de Ringer, pour skparer les deux types d'erythrocytes. Les cellules infect&es peuvent provenir du sang d'un animal porteur du parasite ou de cultures in vitro. L'ophation d'isookmentestrapide et, ainsi qu'on a pu le v6rifier, elle ne compromet pas la viabilitk des parasites et n'affecte pas leurs caract&ristiques ultrastructurelles. En faciltant I'isolement de formes relativement homogines des stadestrythrocytaires asexues dePfalciparum, les proc6&Is mis au point devraient aider les biochimistes et les immu- nologistes dans leurs travaux. REFERENCES 1. GEIMAN, Q. M. & MEAGHER, M. Nature (London), 215: 437-439 (1967). 2. TRAGER, W. & JENSEN, J. B. Science, 193: 673-675 (1976). 3. CHIEN, S. Biophysical behavior of red cells in suspen- sion. In: The red blood cell, 2nd ed., New York, Aca- demic Press, 1975, Vol. 11. pp. 1031-1133. 4. COULSON, A. S. & CHALMERS, D. G. Lancer, 1: 468-469 (1964). 5. PASVOL, G. ETAL Annals of tropical medicine andpara- sitology, 72: 87-88 (1978). 6. LANGRETIH, S. G. ET AL Journal ofprotozoology, 25: 443-452 (1978). 7. TRAGER, W. Expermental parasitology, 8: 265-273 (1959). 8. REESE, R. T. ET AL. Proceedings of the National Academy of Sciences of the United Stales of Amnerica, 75: 5665-5668 (1978).