277 TRANSACTIONSOF THE ROYAL SOCIETYOF TROPICAL MEDICINE AND HYGIENE, VOL. 71, No. 4,1977.
Culture
of malaria parasites G. A. BUTCHER
Guy’s Hospital
Medical
School, London
AND
W. H. G. RICHARDS Wellcome
Research
Laboratories,
Summary A human malaria vaccine that will protect against the blood stages of Plasmodium falciparum is dependent upon the continuous culture of the parasite. Scientists have demonstrated that it is now possible to achieve this on a small scale. Present expertise needs to be expanded to produce larger quantities of material for antigenic studies and potential vaccine production. Introduction A continuous culture of malaria parasites is a prerequisite for the production of parasite antigen in sufficient quantity, of a required standard and potency, and free from biological contamination. Whilst the continuous culture of the asexual parasites appears to be promising, the other stages should not be neglected. Workers investigating the exo-erythrocytic stages have, using avian malaria, been able to infect mouse liver cells in vitro (BEAUDOIN et al., 1974). The human malaria preerythrocytic stage is self-limiting and research into the cultivation of the exo-erythrocytic stages is not sufficiently advanced to assume that work on this aspect of malaria culture will lead to a practical vaccine in the forseeable future. Similarly, the sexual stages are selflimiting. The results achieved by CARTER& CHEN (1976) in using birds immunized with a Plasmodium gallinaceum gametocyte vaccine to prevent the subsequent infection of mosquitoes are encouraging, but a successful human vaccine is not envisaged in the near future. While most of the investigations into the culture of the asexual stages of the malaria parasite have involved development within erythrocytes, the continuous culture of the parasites extracellularly would have advantages. It might be possible to utilize outdated red cells as a source of haemoglobin and it would also reduce the risk of contamination with host cell membrane. Some extracellular development of P. falciparum has been reported by TRAGER (1974); however, work in this area may be of more immediate value in elucidating the biochemical requirements of the parasite. The first recorded attempts to grow the erythrocytic stages of the malaria parasite in vitro were made by BASS & JOHNS in 1912 when, utilizing whole blood supplemented with glucose they were able to observe the development of P. falciparum to the schizont stage. Since then many attempts have been made to maintain the erythrocytic cycle of malaria in red cell culture for a prolonged period (BERTAGNA et al., 1972). Most of these attempts have been with the avian or simian models although rodent and human parasites have not been neglected.
Beckenham,
Kent,
U.K.
Continuous culture was partially successful with the avian P. gallinaceum (ANDERSON,1953), while PHILIPS and his co-workers (1972) were able to maintain P. falciparum through several cycles. Using rodent malarias, parasite multiplication through more than one cycle is rarely achieved (COOMBES & GUTTERIDGE, 1975; SMALLEY & BUTCHER, 1975; WILLIAMS & RICHARDS, 1973). Continuous culture of erythrocytic forms has recently been achieved by two independent groups, TRAGER & JENSEN (1976) and HAYNES et al. (1976) using P. falciparum. TRAGER’S group reported cultivation of the parasite for at least three months, and at the time of writing this has been considerably extended. Two methods of culture have been used, the continuous perfusion of fresh culture medium over a static layer of cells and culture in petri dishes. While the level of parasites present in the culture was low the overall multiplication during the three-month culture period was very high (42s). The HAYNES/DIGGS group (1976) also used static cultures and kept the parasites alive for 22 days until a broken tube prematurely ended the experiment. The two groups developed methods considerably different with respect to media, culture vessels and gas mixtures (Table I). Table I - Plasmodium falciparum culture conditions Rockefeller WRAIR (TRAGER
&JENSEN,
1976) Medium RPM1 1640 Additives Hepes Gas Vessel
18%Oz,3%COz balance N, Petri dish, Constant flow vessel
(HAYNES
et al.,
1976) 199E Tes, 2 mercaptoethanol,Vitamin E, Glucose 6.6 % 02, 3 % co2 balance N, Tissue culture flask (30 ml)
The essential components responsible for success may lie in the use of fully viable parasites from an acute infection of an appropriate strain, the use of human erythrocytes, the maintenance of a constant pH and the persistence of the workers in culturing even when the parasitaemias were extremely low. Of these factors, the use of human red cells may be the most important, as these have a lower metabolic rate and longer life span in vivo than erythrocytes of some other mammalian species (Table II) and they may therefore survive better in vitro. Attempts by one of us (G.A.B.) and others (TRIGG, personal communication) using TRAGER’S methods to culture rhesus monkey erythrocytes infected with P. knowlesi have been much less successful, a week being the maximal length of parasite survival.
278
SYMPOSIUMONPROSPECTS FORMALARIA
Table II - Mammalian erythrocyte metabolism Survival Time Red Blood Glucose Utilization pmoles/hr/ml Days Cell species 1.2-2.1 120 Man 86-133 1.3-3 .o Dog 140-l 50 Horse 0.6 Rabbit 2.95 57 5.6 55 Rat
American
VACCINES Journal
of Tropical
Medicine
and Hygiene,
2,234242. Bass, C. C. & Johns, F. M. (1912). The cultivation of malarial plasmodia (Plasmodium vivax and Plasmodium falciparum) in vitro. Journal of Experimental Medicine, 16, 567-579. Beaudoin, R. L., Strome, C. P. A. & Clutter, W. G. (1974). Cultivation of avian malaria parasites in mammalian liver cells. Experimental Parasitology, 36, 355-359.
It is now possible to culture P. falciparum over considerable periods of time and theoretically the key to production of a successful vaccine is available. There are however a number of problems that need to be studied before malaria vaccine production on any significant scale can be considered. 1. Present methods of continuous culture are on a very small scale and will have to be considerably expanded to give larger volumes of material with higher rates of infected cells. 2. Parasites in long term culture lose their synchronicity (TRAGER & JENSEN,1976) making isolation of specific stages more difficult. 3. For economic reasons, a simplified culture medium is required, as are the ability to use outdated red cells and a substitute for human serum. 4. Nothing is known of the antigenicity of cultured parasites. Do they maintain normal antigens over a long period? This is a prime area for investigation. 5. Isolation of pure preparations of merozoites of P. falciparum has not proved as easy as with P. knowlesi (MITCHELL, 1976 unpublished work). Suitable methods for merozoite isolation and possibly for extracting the relevant antigen(s) need to be developed. 6. The seed material from which cultures are produced has to be shown to be free of biological contaminants, e.g. viruses. 7. Suitable techniques need to be developed to store the P. falciparum merozoite based vaccines while retaining their immunogenicity. References Anderson, C. R. (1953). Continuous propagation of Plasmodium gallinaceum in chicken erythrocytes.
Bertagna, P., Cohen, S., Geiman, Q. M., Haworth, J., Konigk, E., Richards, W. H. G. & Trigg, P. I. (1972). Cultivation techniques for the erythrocytic stages of malaria parasites. Bulletin of the World Health Organisation,
47, 357-373.
Carter, R. & Chen, D. H. (1976). Malaria transmission blocked by immunisation with gametes of the malaria parasite. Nature, 263, 57-60. Coombs, G. H. & Gutteridge, W. E. (1975). Growth in vitro and metabolism of Plasmodium vinckei chabaudi. Journal of Protozoology,
22, 555-560.
Haynes, J. D., Diggs, C. L., Hines, F. A. & Desjardins, R. E. (1976). Culture of human malaria parasites Plasmodium falciparum.
Nature, 263, 767-769.
Mitchell, G. H. (1976). (Unpublished work.) Phillips, R. S., Trigg, P. I., Scott-Finnigan, T. J. & Bartholomew. R. K. (1972). Culture of Plasmodium falciparum in. vitro: a sub-culture technique used for demonstrating anti-plasmodial activity in serum from some Gambians, resident in an endemic malarious area. Parasitology, 65, 525-535. Smalley, M. E. & Butcher, G. A. (1975). The in vitro culture of the blood stages of Plasmodium berghei. International Journal for Parasitology, 5, 131-132. Trager, W. (1974). Initial extracellular development in vitro of Plasmodium falciparum. Proceedings of the Congress Third International 1973. Vol. I, pp. 132-133.
of Parasitology.
Munich
Trager, W. & Jensen, J. B. (1976). Human malaria parasites in continuous culture. Science, 193, 673-675, Trigg, P. I. (Personal communication.) Williams, S. G. & Richards, W. H. G. (1973). Malaria studies in vitro. 1: Techniques for the preparation and culture of leucocyte-free blood-dilution cultures of Plasmodia. Annals of Tropical Medicine and Parasitology, 67, 169-178.
Culture
of malaria parasites G. A. BUTCHER
Guy’s Hospital
Medical
School, London
AND
W. H. G. RICHARDS Wellcome
Research
Laboratories,
Summary A human malaria vaccine that will protect against the blood stages of Plasmodium falciparum is dependent upon the continuous culture of the parasite. Scientists have demonstrated that it is now possible to achieve this on a small scale. Present expertise needs to be expanded to produce larger quantities of material for antigenic studies and potential vaccine production. Introduction A continuous culture of malaria parasites is a prerequisite for the production of parasite antigen in sufficient quantity, of a required standard and potency, and free from biological contamination. Whilst the continuous culture of the asexual parasites appears to be promising, the other stages should not be neglected. Workers investigating the exo-erythrocytic stages have, using avian malaria, been able to infect mouse liver cells in vitro (BEAUDOIN et al., 1974). The human malaria preerythrocytic stage is self-limiting and research into the cultivation of the exo-erythrocytic stages is not sufficiently advanced to assume that work on this aspect of malaria culture will lead to a practical vaccine in the forseeable future. Similarly, the sexual stages are selflimiting. The results achieved by CARTER& CHEN (1976) in using birds immunized with a Plasmodium gallinaceum gametocyte vaccine to prevent the subsequent infection of mosquitoes are encouraging, but a successful human vaccine is not envisaged in the near future. While most of the investigations into the culture of the asexual stages of the malaria parasite have involved development within erythrocytes, the continuous culture of the parasites extracellularly would have advantages. It might be possible to utilize outdated red cells as a source of haemoglobin and it would also reduce the risk of contamination with host cell membrane. Some extracellular development of P. falciparum has been reported by TRAGER (1974); however, work in this area may be of more immediate value in elucidating the biochemical requirements of the parasite. The first recorded attempts to grow the erythrocytic stages of the malaria parasite in vitro were made by BASS & JOHNS in 1912 when, utilizing whole blood supplemented with glucose they were able to observe the development of P. falciparum to the schizont stage. Since then many attempts have been made to maintain the erythrocytic cycle of malaria in red cell culture for a prolonged period (BERTAGNA et al., 1972). Most of these attempts have been with the avian or simian models although rodent and human parasites have not been neglected.
Beckenham,
Kent,
U.K.
Continuous culture was partially successful with the avian P. gallinaceum (ANDERSON,1953), while PHILIPS and his co-workers (1972) were able to maintain P. falciparum through several cycles. Using rodent malarias, parasite multiplication through more than one cycle is rarely achieved (COOMBES & GUTTERIDGE, 1975; SMALLEY & BUTCHER, 1975; WILLIAMS & RICHARDS, 1973). Continuous culture of erythrocytic forms has recently been achieved by two independent groups, TRAGER & JENSEN (1976) and HAYNES et al. (1976) using P. falciparum. TRAGER’S group reported cultivation of the parasite for at least three months, and at the time of writing this has been considerably extended. Two methods of culture have been used, the continuous perfusion of fresh culture medium over a static layer of cells and culture in petri dishes. While the level of parasites present in the culture was low the overall multiplication during the three-month culture period was very high (42s). The HAYNES/DIGGS group (1976) also used static cultures and kept the parasites alive for 22 days until a broken tube prematurely ended the experiment. The two groups developed methods considerably different with respect to media, culture vessels and gas mixtures (Table I). Table I - Plasmodium falciparum culture conditions Rockefeller WRAIR (TRAGER
&JENSEN,
1976) Medium RPM1 1640 Additives Hepes Gas Vessel
18%Oz,3%COz balance N, Petri dish, Constant flow vessel
(HAYNES
et al.,
1976) 199E Tes, 2 mercaptoethanol,Vitamin E, Glucose 6.6 % 02, 3 % co2 balance N, Tissue culture flask (30 ml)
The essential components responsible for success may lie in the use of fully viable parasites from an acute infection of an appropriate strain, the use of human erythrocytes, the maintenance of a constant pH and the persistence of the workers in culturing even when the parasitaemias were extremely low. Of these factors, the use of human red cells may be the most important, as these have a lower metabolic rate and longer life span in vivo than erythrocytes of some other mammalian species (Table II) and they may therefore survive better in vitro. Attempts by one of us (G.A.B.) and others (TRIGG, personal communication) using TRAGER’S methods to culture rhesus monkey erythrocytes infected with P. knowlesi have been much less successful, a week being the maximal length of parasite survival.
278
SYMPOSIUMONPROSPECTS FORMALARIA
Table II - Mammalian erythrocyte metabolism Survival Time Red Blood Glucose Utilization pmoles/hr/ml Days Cell species 1.2-2.1 120 Man 86-133 1.3-3 .o Dog 140-l 50 Horse 0.6 Rabbit 2.95 57 5.6 55 Rat
American
VACCINES Journal
of Tropical
Medicine
and Hygiene,
2,234242. Bass, C. C. & Johns, F. M. (1912). The cultivation of malarial plasmodia (Plasmodium vivax and Plasmodium falciparum) in vitro. Journal of Experimental Medicine, 16, 567-579. Beaudoin, R. L., Strome, C. P. A. & Clutter, W. G. (1974). Cultivation of avian malaria parasites in mammalian liver cells. Experimental Parasitology, 36, 355-359.
It is now possible to culture P. falciparum over considerable periods of time and theoretically the key to production of a successful vaccine is available. There are however a number of problems that need to be studied before malaria vaccine production on any significant scale can be considered. 1. Present methods of continuous culture are on a very small scale and will have to be considerably expanded to give larger volumes of material with higher rates of infected cells. 2. Parasites in long term culture lose their synchronicity (TRAGER & JENSEN,1976) making isolation of specific stages more difficult. 3. For economic reasons, a simplified culture medium is required, as are the ability to use outdated red cells and a substitute for human serum. 4. Nothing is known of the antigenicity of cultured parasites. Do they maintain normal antigens over a long period? This is a prime area for investigation. 5. Isolation of pure preparations of merozoites of P. falciparum has not proved as easy as with P. knowlesi (MITCHELL, 1976 unpublished work). Suitable methods for merozoite isolation and possibly for extracting the relevant antigen(s) need to be developed. 6. The seed material from which cultures are produced has to be shown to be free of biological contaminants, e.g. viruses. 7. Suitable techniques need to be developed to store the P. falciparum merozoite based vaccines while retaining their immunogenicity. References Anderson, C. R. (1953). Continuous propagation of Plasmodium gallinaceum in chicken erythrocytes.
Bertagna, P., Cohen, S., Geiman, Q. M., Haworth, J., Konigk, E., Richards, W. H. G. & Trigg, P. I. (1972). Cultivation techniques for the erythrocytic stages of malaria parasites. Bulletin of the World Health Organisation,
47, 357-373.
Carter, R. & Chen, D. H. (1976). Malaria transmission blocked by immunisation with gametes of the malaria parasite. Nature, 263, 57-60. Coombs, G. H. & Gutteridge, W. E. (1975). Growth in vitro and metabolism of Plasmodium vinckei chabaudi. Journal of Protozoology,
22, 555-560.
Haynes, J. D., Diggs, C. L., Hines, F. A. & Desjardins, R. E. (1976). Culture of human malaria parasites Plasmodium falciparum.
Nature, 263, 767-769.
Mitchell, G. H. (1976). (Unpublished work.) Phillips, R. S., Trigg, P. I., Scott-Finnigan, T. J. & Bartholomew. R. K. (1972). Culture of Plasmodium falciparum in. vitro: a sub-culture technique used for demonstrating anti-plasmodial activity in serum from some Gambians, resident in an endemic malarious area. Parasitology, 65, 525-535. Smalley, M. E. & Butcher, G. A. (1975). The in vitro culture of the blood stages of Plasmodium berghei. International Journal for Parasitology, 5, 131-132. Trager, W. (1974). Initial extracellular development in vitro of Plasmodium falciparum. Proceedings of the Congress Third International 1973. Vol. I, pp. 132-133.
of Parasitology.
Munich
Trager, W. & Jensen, J. B. (1976). Human malaria parasites in continuous culture. Science, 193, 673-675, Trigg, P. I. (Personal communication.) Williams, S. G. & Richards, W. H. G. (1973). Malaria studies in vitro. 1: Techniques for the preparation and culture of leucocyte-free blood-dilution cultures of Plasmodia. Annals of Tropical Medicine and Parasitology, 67, 169-178.
