Proc. Nati. Acad. Sci. USA Vol. 75, No. 11, pp. 5665-5668, November 1978 Immunology
Immunization against malaria with antigen from Plasmodium falciparum cultivated in vitro (immunology/human parasites/genetics/Aotus monkeys)
ROBERT T. REESE*, WILLIAM TRAGER*, JAMES B. JENSEN*, DOROTHY A. MILLERt,
AND RAMANA TANTRAVAHIt *The Rockefeller University, New York, New York 10021; and tDepartment of Human Genetics and Development, College of Physicians and Surgeons, Columbia University, New York, New York 10032 Contributed by William Trager, July 28, 1978
Aotus monkeys, which are generally killed ABSTRACT when infected with the human malaria parasite Plasmodium falciparum, have been identified and grouped by karyotype. These animals were immunized with parasite material obtained from P. falciparum cultivated in vitro which had been maintained in culture for over a year. When sufficient amounts of this antigenic material were used with a synthetic muramyl dipeptide (MDP), protective immunity was induced without presenting the antigen in complete Freund's adjuvant. In 1956 a major international malaria eradication program was initiated by the World Health Organization. This campaign was aimed at elimination of the anopheline mosquito vectors with DDT so that transmission of the disease could be blocked. The initial success was remarkable. Malaria was eliminated from several countries, and on a world basis the number of malaria cases plummeted. As a result of the belief that malaria would soon be eliminated as a world health problem, there was a rapid decline in scientific interest and in drug research. In addition, the economic picture in the developed countries changed so that fewer funds were available for continued malaria eradication. Complicating matters further, the malaria vectors became resistant to DDT and the parasites to drugs. Conditions were therefore ripe for a resurgence, which began in the late 1960s and has now reached frightening proportions (1). Appraisal of the current situation makes it clear that neither insecticides nor antimalaria drugs will be sufficient to control, much less eradicate, the vectors and parasites that are now prevalent. This has renewed interest in the possibility of finding an immunologic solution to the disease. The major pathology caused by malaria is a product of the erythrocytic cycle of the organism. The severe hemolytic anemia induced by the parasite causes stress on the heart, lungs, bone marrow, spleen, and, perhaps most important, the kidneys. Death often closely follows acute renal failure (2). Interruption of the erythrocytic stage of the parasite must therefore be a central target for any protective immune response. Experiments conducted many years ago by Freund et al. (3, 4) demonstrated that formalin-treated erythrocytic stages of Plasmodium lophurae and P. knowlesi were able to induce protective immunity in ducks and monkeys, respectively, if the parasites were administered as an emulsion with mycobacteria in oil. This work was extended by several groups of investigators, who demonstrated that immunization with normal, irradiated, or formalin-treated erythrocytic forms of P. falciparum could be used to modify the normal course of the disease in Aotus monkeys (5-7). The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "ad-
vertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
Merozoites (the infective erythrocytic form of the parasite) have been successfully used to immunize monkeys also. In some of the first studies, it was demonstrated that purified preparations of P. knowlesi merozoites were capable of protecting monkeys if injected in complete Freund's adjuvant (8). During the past year similar studies have been conducted with P. falciparum (9, 10). These recent investigations have shown that parasite material (crude merozoite preparations) isolated from mature P. falciparum schizonts could be used to protectively immunize Aotus monkeys. All of the experiments so far have relied on isolation of parasite material from the blood of parasitized animals (or humans) followed by injection of this material into other animals as an antigen. Some of the problems associated with such a source of antigen are the following: Monkeys infected with malaria generally suffer severe anemias before high levels of parasitemia are achieved. Thus, to obtain significant amounts of parasite material, large numbers of monkeys would be required. Aotus monkeys, which are the most likely candidates for such work, are already in short supply. In addition, it is quite common for an animal that has a severe malaria infection to have an active superinfection with other organisms at the same time. Pooling of material from large numbers of these small animals will increase the probability of contaminants even further. Since it is clear that material to be used for immunization of humans must be free from unnecessary components, it is probable that this can best be achieved by culturing the organism in vitro. With the development of an in vitro procedure for cultivation of P. falciparum, it is now possible to produce large numbers of parasites under controlled conditions in the absence of either leukocytes or other microorganisms (11, 12,t). Thus, a primary object of the present work will be to determine if organisms grown in vitro are sufficiently stable antigenically to provide a suitable antigen source for immunization. The study will also confront the obstacle of proper presentation of the antigen. In all of the work conducted so far it has been essential that the antigen be incorporated in complete Freund's adjuvant to induce significant protection. Because of the undesirable side effects associated with this adjuvant, an alternative means of delivering the antigen must be found. The present investigation will address the probelm of a suitable antigen source, as well as presentation of the antigen in a manner compatible for human use. It will demonstrate that antigen harvested from P. falciparum cultivated in vitro can be used to protectively immunize Aotus monkeys without delivering the antigen in complete Freund's adjuvant. t
Jensen,
(abstr.). 5665
J. B. & Trager, W. (1977) Am. Soc. Trop. Med. Hyg.
5666
Immunology:
Reese et al.
MATERIALS AND METHODS Preparation of Antigen. The FCR-3/FMG strain of P. falciparum was used throughout. It has been maintained in culture continuously for over a year since its isolation from the blood of an individual who contracted malaria in The Gambia. It was chosen because it was not resistant to chloroquine, grew well in culture, and retained its ability to rapidly kill Aotus mon-
keys. Parasitized erythrocytes for culture were often obtained from "flow vial" cultures (11). Petri dish (12) and mechanized "tipper flask" culturest were derived from these. After 6 days in culture, when the parasitemia was greater than 10%, parasitized erythrocytes containing mature trophozoites or schizonts were selectively isolated with Physiogel (13). The final material was composed of 65-90% trophozoites or schizonts, with the remainder being uninfected erythrocytes. Merozoites were harvested by saponin lysis. A concentration of 0.015% saponin (Eastman Kodak, Rochester, NY) was chosen because it would facilitate rupture of segmenters without sufficiently damaging normal erythrocytes so that they would burst as well. Released merozoites were collected after centrifugation for 10 min at 440 X g. Erythrocyte membranes were removed by washing the parasite material in 5% bovine serum albumin (Rekeis Chemical Co., Phoenix, AZ) in a high potassium buffer (14). In this way parasite preparations were obtained that were a mixture of merozoites and throphozoites. A detailed description of the isolation procedure will be presented elsewhere (13). Parasite material isolated by saponin lysis served as the antigen. The number of merozoites in a preparation was estimated by mixing equal volumes of parasite material and human blood. Determination of the number of merozoites (or other parasite forms) relative to the number of erythrocytes provided an estimate of the number of parasites obtained. The antigen was frozen and stored on dry ice if not used the day of harvest. Immunization Procedures. (i) The animals in experiment 1 were immunized with an emulsion formed from equal volumes of antigen and either complete or incomplete Freund's adjuvant (Difco, Detroit, MI). Primary injections were given in the thighs with complete Freund's adjuvant; boosting injections were given in the upper arms with incomplete Freund's adjuvant. Immunizations were spaced 3 weeks apart. (ii) In experiment 2 the monkeys were injected with emulsions prepared from equal volumes of incomplete Freund's adjuvant and antigen. To further enhance responsiveness, we included 170 tig of N-acetylmuramyl-L-alanyl-D-isoglutamine (a synthetic dipeptide, MDP-Pasteur) in the emulsion for each animal. The first two injections were given as in experiment 1; in addition, these animals received a third immunization intraperitoneally. This last injection was composed only of antigen mixed with oil (incomplete Freund's adjuvant). Animals. Aotus sp. monkeys were obtained from Primate Imports (Port Washington, NY). These animals are trapped in the wild in a number of countries in South America. Animals from a single vendor will therefore vary widely in their origins and, thus, their genotypes. To impose some degree of uniformity on animals to be used in an experiment, we karyotyped all animals (15, 16). Chromosome preparations were made in the following manner. Blood was cultured in Panheparin (Abbott Laboratories, N. Chicago, IL). One milliliter of plasma or, more often, whole blood was inoculated into 10 ml of RPMI-1640 (GIBCO, Grand Island, NY) containing 20% heat-inactivated fetal calf serum. A 0.4-ml volume of phytohemagglutinin (GIBCO) was added and the culture was incubated at 370C. During the final hour of incubation, 0.1 ml of Colcemid (10 ,jg/ml, GIBCO) was
Proc. Natl. Acad. Sci. USA 75 (1978)
added to each culture. At 72 hr, cultures were transferred to 15-ml tubes and centrifuged for 7 min at 120 X g. The supernatant was discarded and the cells were suspended in 10 ml of 75 mM KC1 for 10 min at room temperature. The cells were collected by a 7-min centrifugation at 120 X g and fixed in freshly prepared 3:1 (vol/vol) methanol/glacial acetic acid. After an hour the fixative was changed twice and the cells were suspended in 0.5 ml of fresh fixative. The cell suspension was dropped on cold, wet slides which were allowed to air dry. The slides were stored at 4VC until stained. For Q-banding, slides were stained for 7 min in quinacrine-mustard solution, rinsed for 2 min in running tap H20, and mounted in Tris maleate buffer, pH 5.6. The slides were viewed with a fluorescent microscope, and well-spread chromosomes at metaphase were photographed on H and W control film which was developed in H and W developer or in Kodak D-19. The pictures were printed on Ilford paper grade 3 or 4. Chromosomes were counted in every cell. At least two karyotypes were prepared for each animal (16). RESULTS Because genetic background can greatly influence immune responsiveness (17), monkeys were grouped according to karyotype. The monkeys in experiment 1 were all of karyotype III; those in experiment 2 were karyotype II (15, 16). These karyotypes differ only in the presence of one centric fusion chromosome. On the basis of karyotype, both groups of monkeys are believed to be of Colombian origin. To test if the merozoite-containing preparation obtained from cultures could induce protective immunity, we initially injected each of three type III Aotus monkeys with approximately 5 X 108 merozoites. The antigen was emulsified with complete Freund's adjuvant and injected intramuscularly, half into each thigh. Three weeks after the initial immunization, the animals were boosted. To boost, we again injected 5 X 108 merozoites, but this time we delivered the antigen in incomplete Freund's adjuvant. It was divided equally between the two upper arms. Control animals were injected in a similar fashion with the adjuvant emulsified with buffer (14) containing no parasite material. Three weeks after the animals were boosted, they were challenged. Parasitized erythrocytes, which were used for challenge, were obtained from a splenectomized type III monkey that had been infected with the parasite. Only by using such a source (nonfrozen) for challenge can an accurate estimate be made of the parasites to be injected. Donor and recipient animals were matched for karyotype to minimize the likelihood of delivering parasites in an incompatible red blood cell type. Each animal was challenged by intravenous injection of 1 X 106
parasitized erythrocytes. The data are presented in Fig. 1. Two of the controls died only 1 day apart; that is, monkey 83 died on day 15 and monkey 82 died on day 16. The third control, monkey 84 died on day 21. All of the immunized animals suppressed the infection longer than did control monkeys 82 and 83. These data were interpreted to suggest that if larger amounts of antigen were given, complete immunity could be induced. To test this hypothesis, we chose six animals that were of the type II karyotype. Because we believe the amount of antigen injected may well be more important than the way it is presented, the antigen this time was not given in complete Freund's was adjuvant. The vehicle used for presentation of the antigen and Freund's an emulsion containing incomplete adjuvant (18). This dipeptide N-acetylmuramyl-L-alanyl-D-isoglutamine is believed to have the minimal structure requisite for the same type of adjuvant activity provided by Mycobacteria (18, 19).
Immunology:
Proc. Natl. Acad. Sci. USA 75
Reese et al.
0~~~~
a-
X a86 X
-/
2
17
15
13
9
7
5
19
21
23
25
Days af ter infection FIG. 1. Protective immunity induced in Aotus monkeys immunized with antigen in complete Freund's adjuvant. Monkeys 82, 83, and 84 were injected with complete Freund's adjuvant alone (0); monkeys 86, 87, and 88 were injected with complete Freund's adjuvant containing a merozoite-rich fraction of P. falciparum obtained from organisms cultivated in vitro (X). All animals were challenged with 1 X 106 parasitized erythrocytes.
It has also been reported to lack many of the unwanted side effects of complete Freund's adjuvant (19).
Injection intramuscularly into the thighs of control animals
of 170 ,g of N-acetylmuramyl-L-alanyl-D-isoglutamine in an 44
(89)
40 36
32
(85)
(78)
28
24
-
aC, 6
(9X)
oE 12-
8
~~~~~~~~(90)
4 ~~~0
10
12
14
~ 16
18
X
20
- -:x
22
x
24
26
28
30
32
Days after infection FIG. 2. Protective immunity induced in Aotus monkeys immunized with an antigen/muramyl dipeptide mixture emulsified in oil. Monkeys 78, 85, and 89 were injected with the muramyl dipeptide/oil emulsion (0); all were dead by day 17. Monkeys 90, 91; and 92 were injected with a merozoite-rich fraction of P. falciparum ob-
tained from organisms cultivated in vitro emulsified in the same adjuvant (X). Monkey 90 had no detectable parasites after day 30; monkey 91 was dead by day 30; monkey 92 was dead by day 23. All animals had been challenged with 5 X 105 parasitized monkey erythrocytes.
(1978)
5667
emulsion produced some inflammation at the injection site. This was exacerbated by the second injection of 170 ,tg of the dipeptide (in an emulsion) into the upper arms. The second injections induced some swelling of both arms and legs. For this reason, the third injections were given intraperitoneally; the dipeptide was omitted. Each immunized animal received 7 X 108 merozoites on day zero, 5 X 108 merozoites 3 weeks later, and a final 5 X 108 merozoites 6 weeks after the initial immunization. Three weeks after the final injection of parasite material, the animals were challenged with 5 X 105 fresh parasitized erythrocytes. The parasitized cells were introduced into the femoral vein as before. The results of this experiment are found in Fig. 2. All three control monkeys died on day 16 or 17. The parasitemias that occurred in the immunized animals never reached the levels found in the controls. Furthermore, it was approximately a week after the controls died before the first immunized monkey, 92, succumbed. Monkey 91 suppressed its parasitemia until about 2 weeks after the controls had died. The third immunized monkey, 90, never developed a parasitemia that exceeded 0.4%. After day 30, parasites were no longer detectable in its blood. These data clearly demonstrate that Aotus monkeys can be protectively immunized with antigens obtained from P. falciparum that has been grown continuously in culture for over a year. This can be achieved without the need for complete Freund's adjuvant. DISCUSSION For years it has been recognized that immune responsiveness could be genetically controlled (17, 20-24). Some of the earliest work was done by Greenberg and coworkers, who observed that mice of different genetic backgrounds varied in their resistance to infection with P. berghei. Although often overlooked, the work of this group predated by several years the better known studies on immune response regulation conducted with haptens (17, 20-23). Other studies have suggested that human susceptibility to malaria may be linked with HLA type (24). Monkeys of the Aotus species also appear to vary considerably in their responsiveness to infections with P. falciparum. These observations clearly demonstrate that the genetic background of an animal is very important when trying to assess its potenital for mounting a proper immunologic response to malaria. Upon learning approximately a year ago that chromosome studies provide a method by which Aotus monkeys can logically be grouped, we have encouraged use of this procedure for analysis of animals to be used in malaria studies (16). Ma et al. (15, 25) divided the Aotus monkeys into nine karyotypic groups. Three of these groups, including the two used in the present study, live in the same region and have karyotypes that differ only in chromosome number as the result of a centric fusion. However, Aotus from other geographical regions have karyotypes that differ more markedly. Not only can the chromosome number vary among groups of animals, but in some cases the chromosomes themselves differ (15, 16, 25). The Aotus, therefore, includes a group of species, each with the potential of responding in an independent fashion to challenge with P. falciparum. As an example, preliminary data obtained with monkeys of the type VI karotype suggest that these animals are considerably more resistant to infection with P. falciparum than are animals of karyotypes II or III (unpublished data). Thus, to minimize the effects of variability in immune responsiveness, monkeys selected for an experiment were always grouped according to karyotype. Such grouping has generally reduced the amount of variability observed within an experiment. Characterization of animals in this way is also essential if data obtained in different laboratories are to be compared.
5668
Immunology:
Reese et al.
In experiment 1, one of the controls lived significantly longer than the other two. If the response of this animal is considered aberrant, as we believe it should be (all other controls in both experiments died between day 15 and 17), then the data in Fig. 1 suggest that immunization of the animals according to the described procedures does indeed induce some protection. It is likely, however, that the amount of antigen given was simply too small. Experiment 2 differed from the first experiment in three ways. Because we have no control over the types of Aotus that we receive, the second experiment was conducted with animals of a different karyotype (type II). To see if a greater effect could be achieved with a larger amount of antigen, we gave these animals 50% more parasite material than was given the animals in the first experiment. Thus, these animals received a third dose of antigen that contained approximately the same amount of material as was given in each of the previous injections. Finally, the antigen was presented in N-acetylmuramyl-L-alanyl-Disoglutamine (18, 19), a more benign adjuvant than was used in the first experiment. The data presented in Fig. 2 are unequivocal. All of the controls, which received the nonspecific stimulation induced by a mixture of buffer and adjuvant, died within a 24-hr period. In contrast, one of the immunized animals died approximately 1 week after the controls, a second immunized animal died 2 weeks after the controls, and the third immunized animal never displayed a parasitemia exceeding 0.4%. All of the animals suffered a continuous erosion of their hematocrits as the disease progressed. It is this chronic aspect of the disease that finally led to the demise of the two immunized animals that died. Therefore, because of the insidious nature of this type of infection, it is important to allow the disease to run its own course rather than terminating even a low parasitemia with drugs. Premature termination of an infection may deceive one into believing the immune state achieved is greater than that which actually exists. The work presented is thus significant for two major reasons. First, it has demonstrated that parasite material isolated from P. falcAparum that has been cultivated in vitro for over a year can induce protective immunity in monkeys. Second, and equally important, the data make it clear that if sufficient antigen is used together with the synthetic muramyl dipeptide, protective immunity can be induced without the use of complete Freund's adjuvant. We thank E. Lederer for providing the N-acetylmuramyl-L-alanylD-isoglutamine (MDP-Pasteur prepared by Dr. P. Lefrancier at Laboratoires Choay). We also thank Mrs. Greene, Miss Ashton, and Mr.
Proc. Nati. Acad. Sci. USA 75 (1978) Borrero for their help with the work and with preparing the manuscript. This work was supported by Contract ta-C-1373 from the Agency for International Development, Grant T16/181/M2/17(B) from the World Health Organization, and a Grant from the National Foundation-March of Dimes. 1. Peters, W. (1977) N. Engl. J. Med. 297, 1261-1264. 2. Hall, A. P. (1977) Trans. R. Soc. Trop. Med. Hyg. 71, 367379. 3. Freund, J., Sommer, H. W. & Walter, A. W. (1945) Science 102, 200-202. 4. Freund, J., Thomson, K. J., Sommer, H. E., Walter, A. W. & Schenkein, E. L. (1945) Science 102,202-204. 5. Brown, K. N. & Brown, J. N. (1965) Nature (London) 208, 1286-1288. 6. Sadun, E. H., Wellde, B. T. & Hickman, R. L. (1969) Mil. Med. 134, 1165-1175. 7. Voller, A. & Richards, W. H. G. (1968) Lancet ii, 1172-1174. 8. Mitchell, H. G., Butcher, G. A. & Cohen, S. (1975) Immunology 29,397-406. 9. Siddiqui, W. A. (1977) Science 197,388-389. 10. Mitchell, G. H., Richards, W. H. G., Butcher, G. A. & Cohen, S. (1977) Lancet i, 1335-1338. 11. Trager, W. (1978) J. Protozool., in press. 12. Trager, W. & Jensen, J. B. (1976) Science 193, 673-675. 13. Reese, R. T., Langreth, S. G. & Trager, W. (1978) Bull. W. H. O., in press. 14. Trager, W. (1959) Exp. Parasitol. 8,265-273. 15. Ma, N. S. F., Jones, T. C., Miller, A. C., Morgan, L. M. & Adams, E. A. (1976) Lab. Anim. Sci. 26, 1022-1036. 16. Miller, C. K., Miller, D. A., Miller, O. J., Tantravahi, R. & Reese, R. T. (1977) Cytogenet. Cell Genet. 19,215-226. 17. Gasser, D. L. & Silvers, W. K. (1974) in Advances in Immunology, eds. Dixon, F. J. & Kunkel, H. G. (Academic, New York), Vol.
19, pp. 1-66. 18. Ellouz, F., Adam, A., Ciorbaru, R. & Lederer, E. (1974) Biochem. Biophys. Res. Commun. 59,1317-1325. 19. Audibert, F., Chedid, L., Lefrancier, P. & Choay, J. (1976) Cell Immunol. 21, 243-249. 20. Greenberg, J. & Kendrick, L. P. (1957) J. Parasitol. 43, 413419. 21. Greenberg, J. & Kendrick, L. P. (1957) J. Parasitol. 43, 420427. 22. Nadel, E. M., Greenberg, J., Jay, G. E. & Coatney, G. R. (1955) Genetics 40, 620-626. 23. Greenberg, J. & Kendrick, L. (1958) J. Parasitol. 44,492-498. 24. Piazza, A., Belvedere, M. C., Bernoco, D., Conighi, C., Contu, L., Curtoni, E. S., Mattiuz, P. L., Mayr, W., Richiardi, P., Schudeller, G. & Ceppellini, R. (1972) in Histocompatibility Testing 1972, eds. Dausset, J. & Colombani, J. (Williams and Wilkins, Baltimore, MD), pp. 73-84. 25. Ma, N. S. F., Rossan, R. N., Talley, S. T., Harper, J. S., Bebard, M. J. & Jones, T. C. (1978) J. Med. Primatol., in press.
Immunization against malaria with antigen from Plasmodium falciparum cultivated in vitro (immunology/human parasites/genetics/Aotus monkeys)
ROBERT T. REESE*, WILLIAM TRAGER*, JAMES B. JENSEN*, DOROTHY A. MILLERt,
AND RAMANA TANTRAVAHIt *The Rockefeller University, New York, New York 10021; and tDepartment of Human Genetics and Development, College of Physicians and Surgeons, Columbia University, New York, New York 10032 Contributed by William Trager, July 28, 1978
Aotus monkeys, which are generally killed ABSTRACT when infected with the human malaria parasite Plasmodium falciparum, have been identified and grouped by karyotype. These animals were immunized with parasite material obtained from P. falciparum cultivated in vitro which had been maintained in culture for over a year. When sufficient amounts of this antigenic material were used with a synthetic muramyl dipeptide (MDP), protective immunity was induced without presenting the antigen in complete Freund's adjuvant. In 1956 a major international malaria eradication program was initiated by the World Health Organization. This campaign was aimed at elimination of the anopheline mosquito vectors with DDT so that transmission of the disease could be blocked. The initial success was remarkable. Malaria was eliminated from several countries, and on a world basis the number of malaria cases plummeted. As a result of the belief that malaria would soon be eliminated as a world health problem, there was a rapid decline in scientific interest and in drug research. In addition, the economic picture in the developed countries changed so that fewer funds were available for continued malaria eradication. Complicating matters further, the malaria vectors became resistant to DDT and the parasites to drugs. Conditions were therefore ripe for a resurgence, which began in the late 1960s and has now reached frightening proportions (1). Appraisal of the current situation makes it clear that neither insecticides nor antimalaria drugs will be sufficient to control, much less eradicate, the vectors and parasites that are now prevalent. This has renewed interest in the possibility of finding an immunologic solution to the disease. The major pathology caused by malaria is a product of the erythrocytic cycle of the organism. The severe hemolytic anemia induced by the parasite causes stress on the heart, lungs, bone marrow, spleen, and, perhaps most important, the kidneys. Death often closely follows acute renal failure (2). Interruption of the erythrocytic stage of the parasite must therefore be a central target for any protective immune response. Experiments conducted many years ago by Freund et al. (3, 4) demonstrated that formalin-treated erythrocytic stages of Plasmodium lophurae and P. knowlesi were able to induce protective immunity in ducks and monkeys, respectively, if the parasites were administered as an emulsion with mycobacteria in oil. This work was extended by several groups of investigators, who demonstrated that immunization with normal, irradiated, or formalin-treated erythrocytic forms of P. falciparum could be used to modify the normal course of the disease in Aotus monkeys (5-7). The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "ad-
vertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
Merozoites (the infective erythrocytic form of the parasite) have been successfully used to immunize monkeys also. In some of the first studies, it was demonstrated that purified preparations of P. knowlesi merozoites were capable of protecting monkeys if injected in complete Freund's adjuvant (8). During the past year similar studies have been conducted with P. falciparum (9, 10). These recent investigations have shown that parasite material (crude merozoite preparations) isolated from mature P. falciparum schizonts could be used to protectively immunize Aotus monkeys. All of the experiments so far have relied on isolation of parasite material from the blood of parasitized animals (or humans) followed by injection of this material into other animals as an antigen. Some of the problems associated with such a source of antigen are the following: Monkeys infected with malaria generally suffer severe anemias before high levels of parasitemia are achieved. Thus, to obtain significant amounts of parasite material, large numbers of monkeys would be required. Aotus monkeys, which are the most likely candidates for such work, are already in short supply. In addition, it is quite common for an animal that has a severe malaria infection to have an active superinfection with other organisms at the same time. Pooling of material from large numbers of these small animals will increase the probability of contaminants even further. Since it is clear that material to be used for immunization of humans must be free from unnecessary components, it is probable that this can best be achieved by culturing the organism in vitro. With the development of an in vitro procedure for cultivation of P. falciparum, it is now possible to produce large numbers of parasites under controlled conditions in the absence of either leukocytes or other microorganisms (11, 12,t). Thus, a primary object of the present work will be to determine if organisms grown in vitro are sufficiently stable antigenically to provide a suitable antigen source for immunization. The study will also confront the obstacle of proper presentation of the antigen. In all of the work conducted so far it has been essential that the antigen be incorporated in complete Freund's adjuvant to induce significant protection. Because of the undesirable side effects associated with this adjuvant, an alternative means of delivering the antigen must be found. The present investigation will address the probelm of a suitable antigen source, as well as presentation of the antigen in a manner compatible for human use. It will demonstrate that antigen harvested from P. falciparum cultivated in vitro can be used to protectively immunize Aotus monkeys without delivering the antigen in complete Freund's adjuvant. t
Jensen,
(abstr.). 5665
J. B. & Trager, W. (1977) Am. Soc. Trop. Med. Hyg.
5666
Immunology:
Reese et al.
MATERIALS AND METHODS Preparation of Antigen. The FCR-3/FMG strain of P. falciparum was used throughout. It has been maintained in culture continuously for over a year since its isolation from the blood of an individual who contracted malaria in The Gambia. It was chosen because it was not resistant to chloroquine, grew well in culture, and retained its ability to rapidly kill Aotus mon-
keys. Parasitized erythrocytes for culture were often obtained from "flow vial" cultures (11). Petri dish (12) and mechanized "tipper flask" culturest were derived from these. After 6 days in culture, when the parasitemia was greater than 10%, parasitized erythrocytes containing mature trophozoites or schizonts were selectively isolated with Physiogel (13). The final material was composed of 65-90% trophozoites or schizonts, with the remainder being uninfected erythrocytes. Merozoites were harvested by saponin lysis. A concentration of 0.015% saponin (Eastman Kodak, Rochester, NY) was chosen because it would facilitate rupture of segmenters without sufficiently damaging normal erythrocytes so that they would burst as well. Released merozoites were collected after centrifugation for 10 min at 440 X g. Erythrocyte membranes were removed by washing the parasite material in 5% bovine serum albumin (Rekeis Chemical Co., Phoenix, AZ) in a high potassium buffer (14). In this way parasite preparations were obtained that were a mixture of merozoites and throphozoites. A detailed description of the isolation procedure will be presented elsewhere (13). Parasite material isolated by saponin lysis served as the antigen. The number of merozoites in a preparation was estimated by mixing equal volumes of parasite material and human blood. Determination of the number of merozoites (or other parasite forms) relative to the number of erythrocytes provided an estimate of the number of parasites obtained. The antigen was frozen and stored on dry ice if not used the day of harvest. Immunization Procedures. (i) The animals in experiment 1 were immunized with an emulsion formed from equal volumes of antigen and either complete or incomplete Freund's adjuvant (Difco, Detroit, MI). Primary injections were given in the thighs with complete Freund's adjuvant; boosting injections were given in the upper arms with incomplete Freund's adjuvant. Immunizations were spaced 3 weeks apart. (ii) In experiment 2 the monkeys were injected with emulsions prepared from equal volumes of incomplete Freund's adjuvant and antigen. To further enhance responsiveness, we included 170 tig of N-acetylmuramyl-L-alanyl-D-isoglutamine (a synthetic dipeptide, MDP-Pasteur) in the emulsion for each animal. The first two injections were given as in experiment 1; in addition, these animals received a third immunization intraperitoneally. This last injection was composed only of antigen mixed with oil (incomplete Freund's adjuvant). Animals. Aotus sp. monkeys were obtained from Primate Imports (Port Washington, NY). These animals are trapped in the wild in a number of countries in South America. Animals from a single vendor will therefore vary widely in their origins and, thus, their genotypes. To impose some degree of uniformity on animals to be used in an experiment, we karyotyped all animals (15, 16). Chromosome preparations were made in the following manner. Blood was cultured in Panheparin (Abbott Laboratories, N. Chicago, IL). One milliliter of plasma or, more often, whole blood was inoculated into 10 ml of RPMI-1640 (GIBCO, Grand Island, NY) containing 20% heat-inactivated fetal calf serum. A 0.4-ml volume of phytohemagglutinin (GIBCO) was added and the culture was incubated at 370C. During the final hour of incubation, 0.1 ml of Colcemid (10 ,jg/ml, GIBCO) was
Proc. Natl. Acad. Sci. USA 75 (1978)
added to each culture. At 72 hr, cultures were transferred to 15-ml tubes and centrifuged for 7 min at 120 X g. The supernatant was discarded and the cells were suspended in 10 ml of 75 mM KC1 for 10 min at room temperature. The cells were collected by a 7-min centrifugation at 120 X g and fixed in freshly prepared 3:1 (vol/vol) methanol/glacial acetic acid. After an hour the fixative was changed twice and the cells were suspended in 0.5 ml of fresh fixative. The cell suspension was dropped on cold, wet slides which were allowed to air dry. The slides were stored at 4VC until stained. For Q-banding, slides were stained for 7 min in quinacrine-mustard solution, rinsed for 2 min in running tap H20, and mounted in Tris maleate buffer, pH 5.6. The slides were viewed with a fluorescent microscope, and well-spread chromosomes at metaphase were photographed on H and W control film which was developed in H and W developer or in Kodak D-19. The pictures were printed on Ilford paper grade 3 or 4. Chromosomes were counted in every cell. At least two karyotypes were prepared for each animal (16). RESULTS Because genetic background can greatly influence immune responsiveness (17), monkeys were grouped according to karyotype. The monkeys in experiment 1 were all of karyotype III; those in experiment 2 were karyotype II (15, 16). These karyotypes differ only in the presence of one centric fusion chromosome. On the basis of karyotype, both groups of monkeys are believed to be of Colombian origin. To test if the merozoite-containing preparation obtained from cultures could induce protective immunity, we initially injected each of three type III Aotus monkeys with approximately 5 X 108 merozoites. The antigen was emulsified with complete Freund's adjuvant and injected intramuscularly, half into each thigh. Three weeks after the initial immunization, the animals were boosted. To boost, we again injected 5 X 108 merozoites, but this time we delivered the antigen in incomplete Freund's adjuvant. It was divided equally between the two upper arms. Control animals were injected in a similar fashion with the adjuvant emulsified with buffer (14) containing no parasite material. Three weeks after the animals were boosted, they were challenged. Parasitized erythrocytes, which were used for challenge, were obtained from a splenectomized type III monkey that had been infected with the parasite. Only by using such a source (nonfrozen) for challenge can an accurate estimate be made of the parasites to be injected. Donor and recipient animals were matched for karyotype to minimize the likelihood of delivering parasites in an incompatible red blood cell type. Each animal was challenged by intravenous injection of 1 X 106
parasitized erythrocytes. The data are presented in Fig. 1. Two of the controls died only 1 day apart; that is, monkey 83 died on day 15 and monkey 82 died on day 16. The third control, monkey 84 died on day 21. All of the immunized animals suppressed the infection longer than did control monkeys 82 and 83. These data were interpreted to suggest that if larger amounts of antigen were given, complete immunity could be induced. To test this hypothesis, we chose six animals that were of the type II karyotype. Because we believe the amount of antigen injected may well be more important than the way it is presented, the antigen this time was not given in complete Freund's was adjuvant. The vehicle used for presentation of the antigen and Freund's an emulsion containing incomplete adjuvant (18). This dipeptide N-acetylmuramyl-L-alanyl-D-isoglutamine is believed to have the minimal structure requisite for the same type of adjuvant activity provided by Mycobacteria (18, 19).
Immunology:
Proc. Natl. Acad. Sci. USA 75
Reese et al.
0~~~~
a-
X a86 X
-/
2
17
15
13
9
7
5
19
21
23
25
Days af ter infection FIG. 1. Protective immunity induced in Aotus monkeys immunized with antigen in complete Freund's adjuvant. Monkeys 82, 83, and 84 were injected with complete Freund's adjuvant alone (0); monkeys 86, 87, and 88 were injected with complete Freund's adjuvant containing a merozoite-rich fraction of P. falciparum obtained from organisms cultivated in vitro (X). All animals were challenged with 1 X 106 parasitized erythrocytes.
It has also been reported to lack many of the unwanted side effects of complete Freund's adjuvant (19).
Injection intramuscularly into the thighs of control animals
of 170 ,g of N-acetylmuramyl-L-alanyl-D-isoglutamine in an 44
(89)
40 36
32
(85)
(78)
28
24
-
aC, 6
(9X)
oE 12-
8
~~~~~~~~(90)
4 ~~~0
10
12
14
~ 16
18
X
20
- -:x
22
x
24
26
28
30
32
Days after infection FIG. 2. Protective immunity induced in Aotus monkeys immunized with an antigen/muramyl dipeptide mixture emulsified in oil. Monkeys 78, 85, and 89 were injected with the muramyl dipeptide/oil emulsion (0); all were dead by day 17. Monkeys 90, 91; and 92 were injected with a merozoite-rich fraction of P. falciparum ob-
tained from organisms cultivated in vitro emulsified in the same adjuvant (X). Monkey 90 had no detectable parasites after day 30; monkey 91 was dead by day 30; monkey 92 was dead by day 23. All animals had been challenged with 5 X 105 parasitized monkey erythrocytes.
(1978)
5667
emulsion produced some inflammation at the injection site. This was exacerbated by the second injection of 170 ,tg of the dipeptide (in an emulsion) into the upper arms. The second injections induced some swelling of both arms and legs. For this reason, the third injections were given intraperitoneally; the dipeptide was omitted. Each immunized animal received 7 X 108 merozoites on day zero, 5 X 108 merozoites 3 weeks later, and a final 5 X 108 merozoites 6 weeks after the initial immunization. Three weeks after the final injection of parasite material, the animals were challenged with 5 X 105 fresh parasitized erythrocytes. The parasitized cells were introduced into the femoral vein as before. The results of this experiment are found in Fig. 2. All three control monkeys died on day 16 or 17. The parasitemias that occurred in the immunized animals never reached the levels found in the controls. Furthermore, it was approximately a week after the controls died before the first immunized monkey, 92, succumbed. Monkey 91 suppressed its parasitemia until about 2 weeks after the controls had died. The third immunized monkey, 90, never developed a parasitemia that exceeded 0.4%. After day 30, parasites were no longer detectable in its blood. These data clearly demonstrate that Aotus monkeys can be protectively immunized with antigens obtained from P. falciparum that has been grown continuously in culture for over a year. This can be achieved without the need for complete Freund's adjuvant. DISCUSSION For years it has been recognized that immune responsiveness could be genetically controlled (17, 20-24). Some of the earliest work was done by Greenberg and coworkers, who observed that mice of different genetic backgrounds varied in their resistance to infection with P. berghei. Although often overlooked, the work of this group predated by several years the better known studies on immune response regulation conducted with haptens (17, 20-23). Other studies have suggested that human susceptibility to malaria may be linked with HLA type (24). Monkeys of the Aotus species also appear to vary considerably in their responsiveness to infections with P. falciparum. These observations clearly demonstrate that the genetic background of an animal is very important when trying to assess its potenital for mounting a proper immunologic response to malaria. Upon learning approximately a year ago that chromosome studies provide a method by which Aotus monkeys can logically be grouped, we have encouraged use of this procedure for analysis of animals to be used in malaria studies (16). Ma et al. (15, 25) divided the Aotus monkeys into nine karyotypic groups. Three of these groups, including the two used in the present study, live in the same region and have karyotypes that differ only in chromosome number as the result of a centric fusion. However, Aotus from other geographical regions have karyotypes that differ more markedly. Not only can the chromosome number vary among groups of animals, but in some cases the chromosomes themselves differ (15, 16, 25). The Aotus, therefore, includes a group of species, each with the potential of responding in an independent fashion to challenge with P. falciparum. As an example, preliminary data obtained with monkeys of the type VI karotype suggest that these animals are considerably more resistant to infection with P. falciparum than are animals of karyotypes II or III (unpublished data). Thus, to minimize the effects of variability in immune responsiveness, monkeys selected for an experiment were always grouped according to karyotype. Such grouping has generally reduced the amount of variability observed within an experiment. Characterization of animals in this way is also essential if data obtained in different laboratories are to be compared.
5668
Immunology:
Reese et al.
In experiment 1, one of the controls lived significantly longer than the other two. If the response of this animal is considered aberrant, as we believe it should be (all other controls in both experiments died between day 15 and 17), then the data in Fig. 1 suggest that immunization of the animals according to the described procedures does indeed induce some protection. It is likely, however, that the amount of antigen given was simply too small. Experiment 2 differed from the first experiment in three ways. Because we have no control over the types of Aotus that we receive, the second experiment was conducted with animals of a different karyotype (type II). To see if a greater effect could be achieved with a larger amount of antigen, we gave these animals 50% more parasite material than was given the animals in the first experiment. Thus, these animals received a third dose of antigen that contained approximately the same amount of material as was given in each of the previous injections. Finally, the antigen was presented in N-acetylmuramyl-L-alanyl-Disoglutamine (18, 19), a more benign adjuvant than was used in the first experiment. The data presented in Fig. 2 are unequivocal. All of the controls, which received the nonspecific stimulation induced by a mixture of buffer and adjuvant, died within a 24-hr period. In contrast, one of the immunized animals died approximately 1 week after the controls, a second immunized animal died 2 weeks after the controls, and the third immunized animal never displayed a parasitemia exceeding 0.4%. All of the animals suffered a continuous erosion of their hematocrits as the disease progressed. It is this chronic aspect of the disease that finally led to the demise of the two immunized animals that died. Therefore, because of the insidious nature of this type of infection, it is important to allow the disease to run its own course rather than terminating even a low parasitemia with drugs. Premature termination of an infection may deceive one into believing the immune state achieved is greater than that which actually exists. The work presented is thus significant for two major reasons. First, it has demonstrated that parasite material isolated from P. falcAparum that has been cultivated in vitro for over a year can induce protective immunity in monkeys. Second, and equally important, the data make it clear that if sufficient antigen is used together with the synthetic muramyl dipeptide, protective immunity can be induced without the use of complete Freund's adjuvant. We thank E. Lederer for providing the N-acetylmuramyl-L-alanylD-isoglutamine (MDP-Pasteur prepared by Dr. P. Lefrancier at Laboratoires Choay). We also thank Mrs. Greene, Miss Ashton, and Mr.
Proc. Nati. Acad. Sci. USA 75 (1978) Borrero for their help with the work and with preparing the manuscript. This work was supported by Contract ta-C-1373 from the Agency for International Development, Grant T16/181/M2/17(B) from the World Health Organization, and a Grant from the National Foundation-March of Dimes. 1. Peters, W. (1977) N. Engl. J. Med. 297, 1261-1264. 2. Hall, A. P. (1977) Trans. R. Soc. Trop. Med. Hyg. 71, 367379. 3. Freund, J., Sommer, H. W. & Walter, A. W. (1945) Science 102, 200-202. 4. Freund, J., Thomson, K. J., Sommer, H. E., Walter, A. W. & Schenkein, E. L. (1945) Science 102,202-204. 5. Brown, K. N. & Brown, J. N. (1965) Nature (London) 208, 1286-1288. 6. Sadun, E. H., Wellde, B. T. & Hickman, R. L. (1969) Mil. Med. 134, 1165-1175. 7. Voller, A. & Richards, W. H. G. (1968) Lancet ii, 1172-1174. 8. Mitchell, H. G., Butcher, G. A. & Cohen, S. (1975) Immunology 29,397-406. 9. Siddiqui, W. A. (1977) Science 197,388-389. 10. Mitchell, G. H., Richards, W. H. G., Butcher, G. A. & Cohen, S. (1977) Lancet i, 1335-1338. 11. Trager, W. (1978) J. Protozool., in press. 12. Trager, W. & Jensen, J. B. (1976) Science 193, 673-675. 13. Reese, R. T., Langreth, S. G. & Trager, W. (1978) Bull. W. H. O., in press. 14. Trager, W. (1959) Exp. Parasitol. 8,265-273. 15. Ma, N. S. F., Jones, T. C., Miller, A. C., Morgan, L. M. & Adams, E. A. (1976) Lab. Anim. Sci. 26, 1022-1036. 16. Miller, C. K., Miller, D. A., Miller, O. J., Tantravahi, R. & Reese, R. T. (1977) Cytogenet. Cell Genet. 19,215-226. 17. Gasser, D. L. & Silvers, W. K. (1974) in Advances in Immunology, eds. Dixon, F. J. & Kunkel, H. G. (Academic, New York), Vol.
19, pp. 1-66. 18. Ellouz, F., Adam, A., Ciorbaru, R. & Lederer, E. (1974) Biochem. Biophys. Res. Commun. 59,1317-1325. 19. Audibert, F., Chedid, L., Lefrancier, P. & Choay, J. (1976) Cell Immunol. 21, 243-249. 20. Greenberg, J. & Kendrick, L. P. (1957) J. Parasitol. 43, 413419. 21. Greenberg, J. & Kendrick, L. P. (1957) J. Parasitol. 43, 420427. 22. Nadel, E. M., Greenberg, J., Jay, G. E. & Coatney, G. R. (1955) Genetics 40, 620-626. 23. Greenberg, J. & Kendrick, L. (1958) J. Parasitol. 44,492-498. 24. Piazza, A., Belvedere, M. C., Bernoco, D., Conighi, C., Contu, L., Curtoni, E. S., Mattiuz, P. L., Mayr, W., Richiardi, P., Schudeller, G. & Ceppellini, R. (1972) in Histocompatibility Testing 1972, eds. Dausset, J. & Colombani, J. (Williams and Wilkins, Baltimore, MD), pp. 73-84. 25. Ma, N. S. F., Rossan, R. N., Talley, S. T., Harper, J. S., Bebard, M. J. & Jones, T. C. (1978) J. Med. Primatol., in press.
