Parasitology (1975), 71, 183-192 With 1 plate and i figures in the text
183
The release of antigens by Plasmodium falciparum R. J. M. WILSON and R. K. BARTHOLOMEW National Institute for Medical Research, Mill Hill, London N W7 1AA, and MEG Laboratories, Fajara, The Gambia, West Africa {Received 4 February 1975) SUMMARY
Antigens were released in vitro from human red cells infected with Plasmodium falciparum only when parasites reached a late stage of schizogony (probably at rupture of infected red cells during reinvasion). Immune complexes formed if antibody was present. Serum from immune Aotus monkeys opsonized parasite debris from artificially disrupted infected Aotus red cells. INTRODUCTION
We have previously reported our attempts to characterize some of the antigens of P. falciparum and have investigated the precipitating antibodies which they stimulate in man under conditions of natural infection (Wilson, McGregor, Hall, Williams & Bartholomew, 1969; McGregor & Wilson, 1971). No details are known, however, of how these and other malarial antigens present themselves to the host's immune system, although this may have important sequelae in directing the immune response (Unanue, 1972). In this paper, we have tried to establish if antigens leak from infected red cells as the parasites mature. This question relates to the possibility that malarial antigens appear on the surface of infected human red cells as has been suggested both by fluorescent antibody studies (Voller & Bray, 1962) and by the susceptibility of infected red cells in certain monkey malarias to agglutination by antibody (Brown & Brown, 1965). We also wished to test the possibility that selective leakage of S-antigens might account for their presence in the plasma in heavy infections of P. falciparum in man (McGregor, Turner, Williams & Hall, 1968). The results of these experiments led us to ascertain if antigens are released, as well as merozoites, when red cells infected with mature schizonts rupture during the reinvasion phase of the erythrocytic cycle. MATERIALS AND METHODS
Cultures Peripheral blood of Gambian children acutely infected with P. falciparum was mixed with heparinized Ringer (Geiman, Siddiqui & Schnell, 1966) to give a final concentration of 17 units heparin/ml. Blood infected with P. falciparum (Camp strain) was also obtained by cardiac puncture from Aotus monkeys. Heparinized blood was transferred directly to culture flasks or, in some instances, the cells were washed twice with Ringer and made up to the original volume in human AB serum
184
R. J. M. WILSON AND R. K. BARTHOLOMEW
before transfer to culture flasks. Five to 10 ml of blood were mixed in a 21flaskwith 80 ml of 199 medium (Glaxo) supplemented with 5 ml foetal calf serum, 2 ml glucose (10% solution), 3-5 ml sodium bicarbonate (6-6% solution) and 1 ml each of penicillin and streptomycin (stock solutions of 104 units/ml). The medium was equilibrated with 5 % CO2/95 % air and the sealed flask rocked in a waterbath at 37 CC. After 8-12 h, additional glucose and bicarbonate were added to most cultures. Aotus monkey blood infected with P. falciparum was labelled with 3 H-L-methionine as described by Wilson (1974 a). Antigens Cells were recovered from cultures by centrifuging at 600 g for 5 min and were washed twice in M/15 phosphate buffer (PBS) before lyophilization. Cells from cultures which included labelled amino acids were washed 5 times in PBS before freezing at —20 °C. Antigens were extracted from washed cells either by resuspending lyophilized preparations in water or by thawing frozen cells, followed by centrifugation at 600 g for 5 min to remove large particles. Samples of supernatant fluids (20-25 ml) were taken from cultures at intervals and concentrated x 40 or x 80 by negative pressure dialysis. Other tests were carried out with an extract of infected placental blood prepared as described by McGregor, Hall, Williams, Hardy & Turner (1966). Antisera Sera from immune adults living in Manduar village (M) in West Kiang Province of the Gambia were used to detect antigens in culture supematants and extracts of cells. Tests for antibodies to La'-antigen were carried out with sera from 22 malarious children (age range 8 months to 8 years) who attended the Outpatients Clinic at Fajara with initial parasitaemias of 100000 to 750000 P. falciparum parasites per 0.01 ml blood. After anti-malarial treatment, serum was obtained from these children at weekly intervals. A group of 16 Gambian children (aged 8 months to 5 years), who were healthy at the time of examination, provided control sera. Serum was also obtained from normal Aotus monkeys and from animals 2 weeks after they had been immunized by 4 infections with the Camp strain of P. falciparum over a period of 16 months. Gel diffusion These methods, as well as a temperature inactivation test to distinguish various groups of antigens, were described by Wilson, McGregor & Wilson (1973). Titres of various antigens and antibodies were obtained from doubling dilutions. Macrophage cultures Aotus monkey leucocytes were obtained from the buffy coat of heparinized blood which had been centrifuged at 600 g for 5 min. The cells were washed once in Kreb's/glucose solution and transferred to Leighton tubes containing coverslips and medium 199 + 5 % foetal bovine serum. After stationary incubation at 37 °C for 3 days, the coverslips were washed twice in Kreb's/glucose solution to remove
Release of antigens by P. falciparum
185
non-adherent cells. To the resulting monolayers of adherent cells from normal or immune Aotus monkeys (the same animals mentioned above), 200 fi\ of fresh heparinized Aotus plasma or plasma+immune Aotus serum was added per ml of Kreb's/ glucose medium. The cells were then incubated for 1 h at 37 CC. Opsonins were detected by adding to each tube 20 /A of the 600 g supernatant of a lysate of tritiated infected Aotus red cells and incubating for a further 60 min at 37 °C; latex beads 0-8-0-007 ft (Microbio Labs) were also added to demonstrate phagocytic activity by the cells. In other experiments, phagocytosis was inhibited by cooling serum treated monolayers on ice prior to and following addition of antigen + the metabolic inhibitor sodium azide (Nelson, 1969). After incubation with antigen, the coverslips were washed repeatedly with Kreb's/glucose solution and air-dried and fixed in methanol before mounting cell side up in neutral mounting medium (Gurr). Autoradiographs were made as described by Wilson (1974a). The number of latex beads, pigment granules and silver grains in 10 cells and in a similar area of background adjacent to the cells in each experimental group was counted by two observers independently. The assessments were in close agreement.
RESULTS
Release of antigens in vitro Human blood infected with synchronous ring-stage parasites of P. falciparum was cultured in vitro and the medium was sampled when the parasites reached the mature trophozoite stage. The concentrated culture fluid was found to contain S-antigen but not the other antigens that were detected in the infected cells. To determine whether S-antigens present in concentrated culture fluids came from growing parasites or were derived from the infected donor's plasma, we tested the supernatant fluids from cultures of washed, infected red cells (centrifuged twice in M/15 PBS at 600 g prior to culture). As shown in Fig. 1 A, antigen was not recovered from the concentrated culture medium until mature schizonts had formed and some of them had ruptured. The traces of S-antigen found throughout experiment C239 (an exception to the previous statement) were presumed to be derived from the donor's plasma due to insufficient washing. Evidence that antigens were present in the infected red cells throughout the period of culture, but were not released until mature schizonts ruptured, is illustrated in Pig. IB. This also shows that the number of precipitin lines obtained from the extracts of packed infected red cells increased as the parasites matured. Antigen titres also increased as the parasite matured (Fig. 2B). In this experiment, two different antisera from Gambian adults (M368 and M214) were used to assay the antigens; the presence and titre of individual (numbered) antigens is indicated in the figure. It is further shown in Fig. 2 A, that the titre of an S-antigen found in culture fluids throughout an experiment (and therefore presumed to originate from the infected donor's plasma) did not increase until schizogony and rupture of erythrocytes occurred. Antigens belonging to the L, R and S-classes were identified in culture medium supernatants, as illustrated in Fig. 3.
186
R. J. M. WILSON AND R. K. BARTHOLOMEW B C. 253-medium
c. 231-medium T
MT
S D R
MTS S
D R
+A
tI
l
l
1
1
C. 232-medium T MT
T
*
T D MT S S _
I
I
I
F
C. 2S3-RBC
S
i i i r C. 239-medium T Mixed stages S
i i r C. 256-medium MT S
D S
*A ^ I I C. 240-medium r
MT S
C. 256-RBC R
A A 12
18
24
30
36
Hours of culture
45
10
I
I
I
l
15
20
25
30
Hours of culture
Fig. 1A. Supernatant fluids from cultures of washed, human red cells infected with P.falciparum were tested at intervals (arrows) for malarial antigens (each horizontal line indicates the occurrence of an individual antigen). These did not appear until mature schizonts had formed and ruptured. Incomplete removal of donor's plasma which contained an S-antigen is indicated in C-239 (dotted line). B. Antigens could be extracted from infected red cells (RBC) throughout the culture period but did not appear in the medium until schizonts ruptured. Abbreviations: T, trophozoite; MT, mature trophozoite; S, schizont; D, disrupted schizonts; R, reinvasion. (From Wilson, 19746.) Fate of antigens released from infected cells
Red cells infected with mature trophozoites (4-8% infection) were taken from a primary culture, washed and subdivided into 3 fresh 25 ml cultures. One of these contained 0-5 ml of plasma from the child who donated the infected red cells, another culture contained 0-2 ml of plasma from an immune adult. The subcultured parasites were grown until schizonts formed and some reinvasion occurred (final parasite multiplication was x 1-1). After centrifugation at 600 g, the supernatant fluids were concentrated x 100 and were stored at —20 °C until they were thawed and tested for the presence of antigens and antibodies. Plasma added to culture medium without cells was treated similarly as a control. The results in Table 1 show that there was loss of both antigen and antibody from the supernatant of the culture containing parasitized cells and adult plasma; this indicated that immune complexes had formed. There was also loss of antibody in the culture with the donor's plasma, but residual malarial antigens were
Release of antigens by P. falciparum 32-i
C. 230 culture medium
MT
HI
187
(8 m)
S (12 m)
S D
26
36
S D
h 16 H
8 4 2 "
P H P 10
'
22
46
Hours of culture C. 234 RBC
• M 358 E23 M214
B
32-i MT
MT S
f h 16 H 8 4 - 12
i\ m F 12
234
23
ii. 29
Hours of culture
Fig. 2 A. Titres of antigens recovered at intervals from the supernatant fluid of a culture of human red cells infected with Plasmodium falciparum. Antigen no. 1 was an S-antigen. The average number of merozoites (m) is indicated at different stages of schizogony. B. Titre of antigens recovered from red cells infected with Plasmodium falciparum. The cells were harvested at intervals, washed and frozen and thawed to provide a lysate which was tested against sera from two immune African adults (M358 and M214). Individual antigens are numbered 1-6 for serum M358 and a-d for serum M214.
present in the supernatant fluid; under these circumstances soluble immune complexes might have formed but this was not tested. As the outpatients with whom we have worked have fulminating infections requiring prompt treatment at the time of presentation, we have not been able to make similar observations of antibody consumption during schizogony in vivo in man. We have, however, observed that in acutely infected children there is frequently a rise in titre of antibody to the La'-antigen following antimalarial treatment (groups a and b in Fig. 4). This might be a 'rebound' of circulating antibody levels after binding by antigen and absorption in vivo, but it seems more likely that death of drug-treated parasites and their removal from the circulation stimulates antibody production. In support of this hypothesis we found that antibodies to the La'-antigen were detected in undiluted serum in only 6 out of 16 healthy Gambian children, compared to 12 of the 22 children who had recently experienced a severe malarial infection. Comparison of groups a and b in Fig. 4 shows further that the titre of antibody to the La'-antigen fell over the course of a month in 5 out of the 14 malarious children who had increased antibody levels after treatment.
188
R. J. M. WILSON AND R. K. BARTHOLOMEW
Lb (II.1CCI
Fig. 3. Antigens present in supernatant fluids after 36 h (S 36) and 46 h (S 46) of culture were characterized by a heat inactivation test (Wilson et al. 1973). Positive control reactions were set up with an extract of infected placental blood (P) and sera from two immune adults (K601 and K621). An S-antigen detected in the culture fluid was not present in the infected placental blood (from a different donor but L and Lb-antigens, as well as the R'-antigen were common to both antigen sources.)
Table 1. The number of antigens and antibodies recovered from cultures in which reinvasion took place Precipitin lines with culture supernatant Source of Addition of antibody infected cells None + Donor plasma + Adult plasma + Adult plasma — Donor plasma * This antibody was La'.
Antigens 2 2 0
NT NT
Antibodies 0 1* 1* 3 3
NT, not tested.
Uptake of antigen by macrophages It is probable that both soluble and insoluble antigens are released when schizonts lyse to release merozoites and residual cell debris. We have demonstrated opsonins which promote phagocytosis of such cytoplasmic debris in Aotus monkeys immune to P. falciparum infection (PI. 1). In these experiments, infected Aotus red cells, labelled with 3H-methionine, were disrupted by freezing and thawing and the
189
Release of antigens by P. falciparum
i
i
i
r i
i
i
i
r
1 2 3 4 5 0 1 2 3 4 5 Weeks Fig. 4. Titres of antibody to La'-antigen in 22 malarious children (groups a-c and 16 healthy children, group d). The malarious children were treated at time 0 and divided into groups according to whether in the following weeks the level of antibody rose (a), rose and fell (6), the remainder (c). 0 1 2
Table 2. Uptake of latex and Plasmodium falciparum antigen by macrophages in vitro Normal monkey Immune monkey Immune serum
—
36 Uptake latex* 51 105 24 18 Uptake pigment* 4 40 Silver grains* 12 47 * Av. no. particles per cell
44 22 52
supernatant fluid was incubated with monolayers of monkey peripheral blood macrophages in the presence or absence of antibody. The labelled material did not adhere to the cells when phagocytosis was inhibited by incubating the monolayers with antigen and antibody in the presence of sodium azide at 4 °C. At 37 °C, however, in the presence of fresh monkey plasma plus serum from immune monkeys, the uptake of pigment granules and radiolabel by normal monkey macrophages was similar to that of macrophages from immune monkeys (Table 2). It was noted
190
R. J. M. WILSON AND B. K. BARTHOLOMEW
that the pigment granules in the crude lysate of infected red cells which was used as antigen in this experiment, were usually associated with stainable cytoplasmic material which was also radiolabelled (see the macrophage from an immune monkey (PI. 1). Only a small amount of label (which might be regarded as 'soluble' antigen or immune complexes) was associated with other vacuoles in the macrophages. DISCUSSION
Although our experiments are not precise about the timing of antigen release and do not exclude leakage of antigens at a late stage of schizogony, the results suggest that antigens were not released in large amounts from schizont-infected red cells growing in vitro, until they ruptured during reinvasion. I t may be argued that artefactual conditions in the cultures caused cell death at this point and that antigen might not be released in a similar fashion in vivo, but the appearance of S-antigens in the plasma of infected humans is against this view, as is the recovery of various classes of antigen from the plasma of Aotus monkeys heavily infected with P. falciparum (Wilson, unpublished). In other malarial systems there is circumstantial evidence for the release of antigens in vivo at schizogony. Cooper & Fogel (1966) showed cyclical changes in complement components in monkeys infected with P. knowlesi. They suggested that immune complex formation might have occurred when merozoites were released. We have shown here that in immune Aotus monkeys opsonins are present which promote phagocytosis of such cytoplasmic debris as might result from schizogony, e.g. pigment granules. Cohen, Butcher & Crandall (1969) have also referred to the release of a radio-labelled material in cultures of P. knowlesi. This material was both acid and antibody-precipitable and was released slowly during the initial phase of parasite growth but more rapidly at schizogony. Our results suggest that numerous antigens, besides S-antigens, are released into the circulation in cell-free form. One implication of this is that antigenic stimulation of the host occurs by routes other than by macrophage degradation of whole infected red cells or whole parasites. The intravascular release of 'soluble' malarial antigens might interfere with the antibody response to antigen which had been taken up by macrophages, as was observed by Spitznagel & Allison (1970) who administered BSA to mice in both soluble and macrophage-associated forms. Competition for high affinity antibodies might also occur between circulating soluble antigens and important antigens on the parasite or infected red cell surface (Wilson, 19746). To account for the finding that usually only S-antigens are detected in the plasma of malarious Africans, we have suggested that antibody to La and other antigens is present in most individuals living in hyperendemic areas and that combination with these antibodies removes the antigens from the circulation (Wilson et al. 1969). Recent reports of circulating soluble antigen-antibody complexes in malarious humans and Aotus monkeys (Houba & Williams, 1972; Houba, in Wilson, 19746; Houba, personal communication) have yet to show, however, whether antigens other than S-antigens are involved. Antibody responses to
Release of antigens by P. falciparum
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S-antigens are frequently short-lived and many of the antigens in this group seem to be poorly immunogenic (Wilson et al. 1975). While we have demonstrated that neutralization of antibody can occur in vitro, this by itself does not satisfactorily explain the absence of circulating Lb and R'-antigens in vivo as antibodies to these are rarely detected (at least by the gel diffusion test) in malarious communities. More information is needed on the relative amounts of different antigens and their location in infected red cells as well as their rates of catabolism by the host, and their inherent immunogenicity. The possible roles in protective immunity and in the pathology of malaria, of the antigens and antigenic materials demonstrated in the present experiments are clearly of some importance. We thank Dr M. E. Wilson for her histological examinations of the parasites grown in vitro and also Dr I. A. McGregor and his African staff who provided the facilities and helped to collect the material at Fajara. REFERENCES BROWN, K. N. & BBOWN, I. N. (1965). Immunity to malaria: antigenic variation in chronic infections of Plasmodium knowlesi. Nature, London 208, 1286-8. COHEN, S., BUTCHER, G. A. & CRANDALL, R. B. (1969). Action of malarial antibody in vitro. Nature, London 223, 368-71. COOPER, N. R. & FOGEL, B. J . (1966). Complement in acute experimental malaria I I . Alterations in the components of complement. Military Medicine 131, 1180—90. GEIMAN, Q. M., SIDDIQUI, W. A. & SCHNELL, J . V. (1966). Plasma replacement for in vitro
culture of Plasmodium knowlesi. Science 153, 1129-30. HOUBA, V. & WILLIAMS, A. I. O. (1972). Soluble serum antigens of P'. falciparum in Nigerians. II. Immunochemical studies. African Journal of Medical Science 3, 309—17. MCGREGOR, I. A., HALL, P . J., WILLIAMS, K., H A R D Y , C. L. S. & TURNER, M. W. (1966).
Demonstration of circulating antibodies to Plasmodium falciparum by gel diffusion techniques. Nature, London 210, 1384-6. MCGREGOR, I . A., TURNER, M. W., WILLIAMS, K. & HALL, P . (1968). Soluble antigens in t h e
blood of African patients with severe Plasmodium falciparum malaria. Lancet i, 881-4. MCGREGOR, I. A. & WILSON, R. J . M. (1971). Precipitating antibodies and immunoglobulins in P . falciparum infections in the Gambia, West Africa. Transactions of the Royal Society of Tropical Medicine and Hygiene 65, 136-45. NELSON, D. S. (1969). Macrophages and Immunity, pp. 94-137. Amsterdam: North-Holland Publishing Co. SPITZNAGEL, J . K. & ALLISON, A. C. (1970). Mode of action of adjuvants: effects on antibody responses to macrophage-associated bovine serum albumin. Journal of Immunology 104, 128-39. UNANUE, E. R. (1972). The regulatory role of macrophages in antigenic stimulation. Advances in Immunology 15, 95-165. VOLLER, A. & BRAY, R. S. (1962). Fluorescent antibody staining as a measure of malarial antibody. Proceedings of the Society of Experimental Biology and Medicine 110, 907-10. WILSON, R. J . M. (1974a). The production of antigens by Plasmodium falciparum in vitro. International Journal of Parasitology 4, 537-47. WILSON, R. J . M. (19746). Soluble antigens as blocking antigens. Ciba Foundation Symposium 25 (new series), pp. 185-203. Associated Scientific Publishers: Amsterdam. WILSON, R. J . M., MCGREGOR, I. A., H A L L , P . WILLIAMS, K., & BARTHOLOMEW, R. (1969).
Antigens associated with Plasmodium falciparum infections in man. Lancet ii, 201-5. WILSON, R. J . M., MCGREGOR, I. A. & WILSON, M. E . (1973). The stability and fractionation
of malarial antigens from the blood of Africans infected with Plasmodium International Journal of Parasitology 3, 511-20.
falciparum.
WILSON, R. J . M., MCGREGOR, I. A., H A L L , P., WILLIAMS, K. & BARTHOLOMEW, R. (1975).
Precipitating antibody response to malarial S-antigens. Transactions of the Royal Society of Tropical Medicine and Hygiene. (In the Press.)
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E. J. M. WILSOW AlfD B. K. BARTHOLOMEW
EXPLANATION OJF PIATB Monolayers of macrophages from normal Aotus monkeys were compared with those from aninaals immune to Plasmodium faldparum in their capacity to phagocytose latex particles and disrupted infected red cell material in the presence or absence of antibody from immune monkeys.
Printed in Great Britain
Parasitology,
Plate 1
Vol. 7 1 , Part 2
Aotus
Monkey Normal
Normal
Macrophages Immune
Pigment granule
Latex
Latex
Immune Aotus serum
R. J. M. WILSOX AXD R. K. BARTHOLOMEW
(Facing p. 192)
183
The release of antigens by Plasmodium falciparum R. J. M. WILSON and R. K. BARTHOLOMEW National Institute for Medical Research, Mill Hill, London N W7 1AA, and MEG Laboratories, Fajara, The Gambia, West Africa {Received 4 February 1975) SUMMARY
Antigens were released in vitro from human red cells infected with Plasmodium falciparum only when parasites reached a late stage of schizogony (probably at rupture of infected red cells during reinvasion). Immune complexes formed if antibody was present. Serum from immune Aotus monkeys opsonized parasite debris from artificially disrupted infected Aotus red cells. INTRODUCTION
We have previously reported our attempts to characterize some of the antigens of P. falciparum and have investigated the precipitating antibodies which they stimulate in man under conditions of natural infection (Wilson, McGregor, Hall, Williams & Bartholomew, 1969; McGregor & Wilson, 1971). No details are known, however, of how these and other malarial antigens present themselves to the host's immune system, although this may have important sequelae in directing the immune response (Unanue, 1972). In this paper, we have tried to establish if antigens leak from infected red cells as the parasites mature. This question relates to the possibility that malarial antigens appear on the surface of infected human red cells as has been suggested both by fluorescent antibody studies (Voller & Bray, 1962) and by the susceptibility of infected red cells in certain monkey malarias to agglutination by antibody (Brown & Brown, 1965). We also wished to test the possibility that selective leakage of S-antigens might account for their presence in the plasma in heavy infections of P. falciparum in man (McGregor, Turner, Williams & Hall, 1968). The results of these experiments led us to ascertain if antigens are released, as well as merozoites, when red cells infected with mature schizonts rupture during the reinvasion phase of the erythrocytic cycle. MATERIALS AND METHODS
Cultures Peripheral blood of Gambian children acutely infected with P. falciparum was mixed with heparinized Ringer (Geiman, Siddiqui & Schnell, 1966) to give a final concentration of 17 units heparin/ml. Blood infected with P. falciparum (Camp strain) was also obtained by cardiac puncture from Aotus monkeys. Heparinized blood was transferred directly to culture flasks or, in some instances, the cells were washed twice with Ringer and made up to the original volume in human AB serum
184
R. J. M. WILSON AND R. K. BARTHOLOMEW
before transfer to culture flasks. Five to 10 ml of blood were mixed in a 21flaskwith 80 ml of 199 medium (Glaxo) supplemented with 5 ml foetal calf serum, 2 ml glucose (10% solution), 3-5 ml sodium bicarbonate (6-6% solution) and 1 ml each of penicillin and streptomycin (stock solutions of 104 units/ml). The medium was equilibrated with 5 % CO2/95 % air and the sealed flask rocked in a waterbath at 37 CC. After 8-12 h, additional glucose and bicarbonate were added to most cultures. Aotus monkey blood infected with P. falciparum was labelled with 3 H-L-methionine as described by Wilson (1974 a). Antigens Cells were recovered from cultures by centrifuging at 600 g for 5 min and were washed twice in M/15 phosphate buffer (PBS) before lyophilization. Cells from cultures which included labelled amino acids were washed 5 times in PBS before freezing at —20 °C. Antigens were extracted from washed cells either by resuspending lyophilized preparations in water or by thawing frozen cells, followed by centrifugation at 600 g for 5 min to remove large particles. Samples of supernatant fluids (20-25 ml) were taken from cultures at intervals and concentrated x 40 or x 80 by negative pressure dialysis. Other tests were carried out with an extract of infected placental blood prepared as described by McGregor, Hall, Williams, Hardy & Turner (1966). Antisera Sera from immune adults living in Manduar village (M) in West Kiang Province of the Gambia were used to detect antigens in culture supematants and extracts of cells. Tests for antibodies to La'-antigen were carried out with sera from 22 malarious children (age range 8 months to 8 years) who attended the Outpatients Clinic at Fajara with initial parasitaemias of 100000 to 750000 P. falciparum parasites per 0.01 ml blood. After anti-malarial treatment, serum was obtained from these children at weekly intervals. A group of 16 Gambian children (aged 8 months to 5 years), who were healthy at the time of examination, provided control sera. Serum was also obtained from normal Aotus monkeys and from animals 2 weeks after they had been immunized by 4 infections with the Camp strain of P. falciparum over a period of 16 months. Gel diffusion These methods, as well as a temperature inactivation test to distinguish various groups of antigens, were described by Wilson, McGregor & Wilson (1973). Titres of various antigens and antibodies were obtained from doubling dilutions. Macrophage cultures Aotus monkey leucocytes were obtained from the buffy coat of heparinized blood which had been centrifuged at 600 g for 5 min. The cells were washed once in Kreb's/glucose solution and transferred to Leighton tubes containing coverslips and medium 199 + 5 % foetal bovine serum. After stationary incubation at 37 °C for 3 days, the coverslips were washed twice in Kreb's/glucose solution to remove
Release of antigens by P. falciparum
185
non-adherent cells. To the resulting monolayers of adherent cells from normal or immune Aotus monkeys (the same animals mentioned above), 200 fi\ of fresh heparinized Aotus plasma or plasma+immune Aotus serum was added per ml of Kreb's/ glucose medium. The cells were then incubated for 1 h at 37 CC. Opsonins were detected by adding to each tube 20 /A of the 600 g supernatant of a lysate of tritiated infected Aotus red cells and incubating for a further 60 min at 37 °C; latex beads 0-8-0-007 ft (Microbio Labs) were also added to demonstrate phagocytic activity by the cells. In other experiments, phagocytosis was inhibited by cooling serum treated monolayers on ice prior to and following addition of antigen + the metabolic inhibitor sodium azide (Nelson, 1969). After incubation with antigen, the coverslips were washed repeatedly with Kreb's/glucose solution and air-dried and fixed in methanol before mounting cell side up in neutral mounting medium (Gurr). Autoradiographs were made as described by Wilson (1974a). The number of latex beads, pigment granules and silver grains in 10 cells and in a similar area of background adjacent to the cells in each experimental group was counted by two observers independently. The assessments were in close agreement.
RESULTS
Release of antigens in vitro Human blood infected with synchronous ring-stage parasites of P. falciparum was cultured in vitro and the medium was sampled when the parasites reached the mature trophozoite stage. The concentrated culture fluid was found to contain S-antigen but not the other antigens that were detected in the infected cells. To determine whether S-antigens present in concentrated culture fluids came from growing parasites or were derived from the infected donor's plasma, we tested the supernatant fluids from cultures of washed, infected red cells (centrifuged twice in M/15 PBS at 600 g prior to culture). As shown in Fig. 1 A, antigen was not recovered from the concentrated culture medium until mature schizonts had formed and some of them had ruptured. The traces of S-antigen found throughout experiment C239 (an exception to the previous statement) were presumed to be derived from the donor's plasma due to insufficient washing. Evidence that antigens were present in the infected red cells throughout the period of culture, but were not released until mature schizonts ruptured, is illustrated in Pig. IB. This also shows that the number of precipitin lines obtained from the extracts of packed infected red cells increased as the parasites matured. Antigen titres also increased as the parasite matured (Fig. 2B). In this experiment, two different antisera from Gambian adults (M368 and M214) were used to assay the antigens; the presence and titre of individual (numbered) antigens is indicated in the figure. It is further shown in Fig. 2 A, that the titre of an S-antigen found in culture fluids throughout an experiment (and therefore presumed to originate from the infected donor's plasma) did not increase until schizogony and rupture of erythrocytes occurred. Antigens belonging to the L, R and S-classes were identified in culture medium supernatants, as illustrated in Fig. 3.
186
R. J. M. WILSON AND R. K. BARTHOLOMEW B C. 253-medium
c. 231-medium T
MT
S D R
MTS S
D R
+A
tI
l
l
1
1
C. 232-medium T MT
T
*
T D MT S S _
I
I
I
F
C. 2S3-RBC
S
i i i r C. 239-medium T Mixed stages S
i i r C. 256-medium MT S
D S
*A ^ I I C. 240-medium r
MT S
C. 256-RBC R
A A 12
18
24
30
36
Hours of culture
45
10
I
I
I
l
15
20
25
30
Hours of culture
Fig. 1A. Supernatant fluids from cultures of washed, human red cells infected with P.falciparum were tested at intervals (arrows) for malarial antigens (each horizontal line indicates the occurrence of an individual antigen). These did not appear until mature schizonts had formed and ruptured. Incomplete removal of donor's plasma which contained an S-antigen is indicated in C-239 (dotted line). B. Antigens could be extracted from infected red cells (RBC) throughout the culture period but did not appear in the medium until schizonts ruptured. Abbreviations: T, trophozoite; MT, mature trophozoite; S, schizont; D, disrupted schizonts; R, reinvasion. (From Wilson, 19746.) Fate of antigens released from infected cells
Red cells infected with mature trophozoites (4-8% infection) were taken from a primary culture, washed and subdivided into 3 fresh 25 ml cultures. One of these contained 0-5 ml of plasma from the child who donated the infected red cells, another culture contained 0-2 ml of plasma from an immune adult. The subcultured parasites were grown until schizonts formed and some reinvasion occurred (final parasite multiplication was x 1-1). After centrifugation at 600 g, the supernatant fluids were concentrated x 100 and were stored at —20 °C until they were thawed and tested for the presence of antigens and antibodies. Plasma added to culture medium without cells was treated similarly as a control. The results in Table 1 show that there was loss of both antigen and antibody from the supernatant of the culture containing parasitized cells and adult plasma; this indicated that immune complexes had formed. There was also loss of antibody in the culture with the donor's plasma, but residual malarial antigens were
Release of antigens by P. falciparum 32-i
C. 230 culture medium
MT
HI
187
(8 m)
S (12 m)
S D
26
36
S D
h 16 H
8 4 2 "
P H P 10
'
22
46
Hours of culture C. 234 RBC
• M 358 E23 M214
B
32-i MT
MT S
f h 16 H 8 4 - 12
i\ m F 12
234
23
ii. 29
Hours of culture
Fig. 2 A. Titres of antigens recovered at intervals from the supernatant fluid of a culture of human red cells infected with Plasmodium falciparum. Antigen no. 1 was an S-antigen. The average number of merozoites (m) is indicated at different stages of schizogony. B. Titre of antigens recovered from red cells infected with Plasmodium falciparum. The cells were harvested at intervals, washed and frozen and thawed to provide a lysate which was tested against sera from two immune African adults (M358 and M214). Individual antigens are numbered 1-6 for serum M358 and a-d for serum M214.
present in the supernatant fluid; under these circumstances soluble immune complexes might have formed but this was not tested. As the outpatients with whom we have worked have fulminating infections requiring prompt treatment at the time of presentation, we have not been able to make similar observations of antibody consumption during schizogony in vivo in man. We have, however, observed that in acutely infected children there is frequently a rise in titre of antibody to the La'-antigen following antimalarial treatment (groups a and b in Fig. 4). This might be a 'rebound' of circulating antibody levels after binding by antigen and absorption in vivo, but it seems more likely that death of drug-treated parasites and their removal from the circulation stimulates antibody production. In support of this hypothesis we found that antibodies to the La'-antigen were detected in undiluted serum in only 6 out of 16 healthy Gambian children, compared to 12 of the 22 children who had recently experienced a severe malarial infection. Comparison of groups a and b in Fig. 4 shows further that the titre of antibody to the La'-antigen fell over the course of a month in 5 out of the 14 malarious children who had increased antibody levels after treatment.
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Lb (II.1CCI
Fig. 3. Antigens present in supernatant fluids after 36 h (S 36) and 46 h (S 46) of culture were characterized by a heat inactivation test (Wilson et al. 1973). Positive control reactions were set up with an extract of infected placental blood (P) and sera from two immune adults (K601 and K621). An S-antigen detected in the culture fluid was not present in the infected placental blood (from a different donor but L and Lb-antigens, as well as the R'-antigen were common to both antigen sources.)
Table 1. The number of antigens and antibodies recovered from cultures in which reinvasion took place Precipitin lines with culture supernatant Source of Addition of antibody infected cells None + Donor plasma + Adult plasma + Adult plasma — Donor plasma * This antibody was La'.
Antigens 2 2 0
NT NT
Antibodies 0 1* 1* 3 3
NT, not tested.
Uptake of antigen by macrophages It is probable that both soluble and insoluble antigens are released when schizonts lyse to release merozoites and residual cell debris. We have demonstrated opsonins which promote phagocytosis of such cytoplasmic debris in Aotus monkeys immune to P. falciparum infection (PI. 1). In these experiments, infected Aotus red cells, labelled with 3H-methionine, were disrupted by freezing and thawing and the
189
Release of antigens by P. falciparum
i
i
i
r i
i
i
i
r
1 2 3 4 5 0 1 2 3 4 5 Weeks Fig. 4. Titres of antibody to La'-antigen in 22 malarious children (groups a-c and 16 healthy children, group d). The malarious children were treated at time 0 and divided into groups according to whether in the following weeks the level of antibody rose (a), rose and fell (6), the remainder (c). 0 1 2
Table 2. Uptake of latex and Plasmodium falciparum antigen by macrophages in vitro Normal monkey Immune monkey Immune serum
—
36 Uptake latex* 51 105 24 18 Uptake pigment* 4 40 Silver grains* 12 47 * Av. no. particles per cell
44 22 52
supernatant fluid was incubated with monolayers of monkey peripheral blood macrophages in the presence or absence of antibody. The labelled material did not adhere to the cells when phagocytosis was inhibited by incubating the monolayers with antigen and antibody in the presence of sodium azide at 4 °C. At 37 °C, however, in the presence of fresh monkey plasma plus serum from immune monkeys, the uptake of pigment granules and radiolabel by normal monkey macrophages was similar to that of macrophages from immune monkeys (Table 2). It was noted
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that the pigment granules in the crude lysate of infected red cells which was used as antigen in this experiment, were usually associated with stainable cytoplasmic material which was also radiolabelled (see the macrophage from an immune monkey (PI. 1). Only a small amount of label (which might be regarded as 'soluble' antigen or immune complexes) was associated with other vacuoles in the macrophages. DISCUSSION
Although our experiments are not precise about the timing of antigen release and do not exclude leakage of antigens at a late stage of schizogony, the results suggest that antigens were not released in large amounts from schizont-infected red cells growing in vitro, until they ruptured during reinvasion. I t may be argued that artefactual conditions in the cultures caused cell death at this point and that antigen might not be released in a similar fashion in vivo, but the appearance of S-antigens in the plasma of infected humans is against this view, as is the recovery of various classes of antigen from the plasma of Aotus monkeys heavily infected with P. falciparum (Wilson, unpublished). In other malarial systems there is circumstantial evidence for the release of antigens in vivo at schizogony. Cooper & Fogel (1966) showed cyclical changes in complement components in monkeys infected with P. knowlesi. They suggested that immune complex formation might have occurred when merozoites were released. We have shown here that in immune Aotus monkeys opsonins are present which promote phagocytosis of such cytoplasmic debris as might result from schizogony, e.g. pigment granules. Cohen, Butcher & Crandall (1969) have also referred to the release of a radio-labelled material in cultures of P. knowlesi. This material was both acid and antibody-precipitable and was released slowly during the initial phase of parasite growth but more rapidly at schizogony. Our results suggest that numerous antigens, besides S-antigens, are released into the circulation in cell-free form. One implication of this is that antigenic stimulation of the host occurs by routes other than by macrophage degradation of whole infected red cells or whole parasites. The intravascular release of 'soluble' malarial antigens might interfere with the antibody response to antigen which had been taken up by macrophages, as was observed by Spitznagel & Allison (1970) who administered BSA to mice in both soluble and macrophage-associated forms. Competition for high affinity antibodies might also occur between circulating soluble antigens and important antigens on the parasite or infected red cell surface (Wilson, 19746). To account for the finding that usually only S-antigens are detected in the plasma of malarious Africans, we have suggested that antibody to La and other antigens is present in most individuals living in hyperendemic areas and that combination with these antibodies removes the antigens from the circulation (Wilson et al. 1969). Recent reports of circulating soluble antigen-antibody complexes in malarious humans and Aotus monkeys (Houba & Williams, 1972; Houba, in Wilson, 19746; Houba, personal communication) have yet to show, however, whether antigens other than S-antigens are involved. Antibody responses to
Release of antigens by P. falciparum
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S-antigens are frequently short-lived and many of the antigens in this group seem to be poorly immunogenic (Wilson et al. 1975). While we have demonstrated that neutralization of antibody can occur in vitro, this by itself does not satisfactorily explain the absence of circulating Lb and R'-antigens in vivo as antibodies to these are rarely detected (at least by the gel diffusion test) in malarious communities. More information is needed on the relative amounts of different antigens and their location in infected red cells as well as their rates of catabolism by the host, and their inherent immunogenicity. The possible roles in protective immunity and in the pathology of malaria, of the antigens and antigenic materials demonstrated in the present experiments are clearly of some importance. We thank Dr M. E. Wilson for her histological examinations of the parasites grown in vitro and also Dr I. A. McGregor and his African staff who provided the facilities and helped to collect the material at Fajara. REFERENCES BROWN, K. N. & BBOWN, I. N. (1965). Immunity to malaria: antigenic variation in chronic infections of Plasmodium knowlesi. Nature, London 208, 1286-8. COHEN, S., BUTCHER, G. A. & CRANDALL, R. B. (1969). Action of malarial antibody in vitro. Nature, London 223, 368-71. COOPER, N. R. & FOGEL, B. J . (1966). Complement in acute experimental malaria I I . Alterations in the components of complement. Military Medicine 131, 1180—90. GEIMAN, Q. M., SIDDIQUI, W. A. & SCHNELL, J . V. (1966). Plasma replacement for in vitro
culture of Plasmodium knowlesi. Science 153, 1129-30. HOUBA, V. & WILLIAMS, A. I. O. (1972). Soluble serum antigens of P'. falciparum in Nigerians. II. Immunochemical studies. African Journal of Medical Science 3, 309—17. MCGREGOR, I. A., HALL, P . J., WILLIAMS, K., H A R D Y , C. L. S. & TURNER, M. W. (1966).
Demonstration of circulating antibodies to Plasmodium falciparum by gel diffusion techniques. Nature, London 210, 1384-6. MCGREGOR, I . A., TURNER, M. W., WILLIAMS, K. & HALL, P . (1968). Soluble antigens in t h e
blood of African patients with severe Plasmodium falciparum malaria. Lancet i, 881-4. MCGREGOR, I. A. & WILSON, R. J . M. (1971). Precipitating antibodies and immunoglobulins in P . falciparum infections in the Gambia, West Africa. Transactions of the Royal Society of Tropical Medicine and Hygiene 65, 136-45. NELSON, D. S. (1969). Macrophages and Immunity, pp. 94-137. Amsterdam: North-Holland Publishing Co. SPITZNAGEL, J . K. & ALLISON, A. C. (1970). Mode of action of adjuvants: effects on antibody responses to macrophage-associated bovine serum albumin. Journal of Immunology 104, 128-39. UNANUE, E. R. (1972). The regulatory role of macrophages in antigenic stimulation. Advances in Immunology 15, 95-165. VOLLER, A. & BRAY, R. S. (1962). Fluorescent antibody staining as a measure of malarial antibody. Proceedings of the Society of Experimental Biology and Medicine 110, 907-10. WILSON, R. J . M. (1974a). The production of antigens by Plasmodium falciparum in vitro. International Journal of Parasitology 4, 537-47. WILSON, R. J . M. (19746). Soluble antigens as blocking antigens. Ciba Foundation Symposium 25 (new series), pp. 185-203. Associated Scientific Publishers: Amsterdam. WILSON, R. J . M., MCGREGOR, I. A., H A L L , P . WILLIAMS, K., & BARTHOLOMEW, R. (1969).
Antigens associated with Plasmodium falciparum infections in man. Lancet ii, 201-5. WILSON, R. J . M., MCGREGOR, I. A. & WILSON, M. E . (1973). The stability and fractionation
of malarial antigens from the blood of Africans infected with Plasmodium International Journal of Parasitology 3, 511-20.
falciparum.
WILSON, R. J . M., MCGREGOR, I. A., H A L L , P., WILLIAMS, K. & BARTHOLOMEW, R. (1975).
Precipitating antibody response to malarial S-antigens. Transactions of the Royal Society of Tropical Medicine and Hygiene. (In the Press.)
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EXPLANATION OJF PIATB Monolayers of macrophages from normal Aotus monkeys were compared with those from aninaals immune to Plasmodium faldparum in their capacity to phagocytose latex particles and disrupted infected red cell material in the presence or absence of antibody from immune monkeys.
Printed in Great Britain
Parasitology,
Plate 1
Vol. 7 1 , Part 2
Aotus
Monkey Normal
Normal
Macrophages Immune
Pigment granule
Latex
Latex
Immune Aotus serum
R. J. M. WILSOX AXD R. K. BARTHOLOMEW
(Facing p. 192)
