Parasite Immunology 1983,5,225-236

Immunization of owl monkeys to Plasmodium falciparum with merozoites from cultures of a knobless clone

W.TRAGER, H.N.LANNERS, H.A.STANLEY* & S.G.LANGRETHt Parasitology Laboratory, The Rockefeller University, New York, N Y 10021, USA

Accepted for publication 22 June 1982

Summary Aotus trivirgatus monkeys of karyotype 2 were treated as follows. Three received two injections of purified merozoites of a knobless (K-) clone of Plasmodium falciparum with muramyl dipeptide as adjuvant; three received similar injections but with merozoites of a wild-type knobby (K+) strain; three controls received MDP with human erythrocytes in the amounts estimated to be present as contaminants in the merozoite preparations. A month after the second injection all nine monkeys were inoculated with parasites of the wild-type knobby strain from another infected Aotus. The monkeys that had received the K- material developed only extremely low infections markedly different from the infections in the controls. Of those that received K + material, one died early with low parasitaemia, one was protected and one had the same level of infection as the controls. In the combined group of immunized animals, four out of six were protected.

Keywords: malaria, Aofus, immunization

Introduction

Recent studies on immunity to malaria, and the possibilities for development of a vaccine, have been summarized in several reviews (Cohen 1979, Desowitz & Miller 1980, Hommel 1981, Wernsdorfer 1981). It is clear that owl monkeys (Aotus trivirgatus) can be immunized to the erythrocytic stages of the major human malarial parasite Plasmodium falciparum by the injection of parasite material mixed either with Freund’s complete adjuvant (Mitchell et al. 1977, Siddiqui 1977) or with muramyl dipeptide (MDP) in mineral oil (Siddiqui et al. 1978, 1979, Reese et al. 1978). The parasite material was obtained either from human cases (Mitchell et al. 1977), infected Aotus monkeys (Siddiqui 1977) or from continuous culture in human erythrocytes (Reese et al. 1978). It is important to note that use of parasites from Aotus to vaccinate Aotus has the advantage of

* Present Address: Research Institute of Scripps Clinic, La Jolla, CA 92037, USA. t Present Address: Department of Microbiology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814, USA.

0141-9838/83/05004225S02.00 0 1983 Blackwell Scientific Publications 225

226 W. Trager et al.

providing a homologous system. On the other hand, it is difficult in practice to prepare large amounts of parasite material from Aotus, whereas these can be readily prepared from large-scale cultures in human erythrocytes (Trager & Jensen 1980). The disadvan- tage of having to use a heterologous system can then be overcome, at least in part, by preparing parasite material as free as possible from host erythrocyte contaminants. Since merozoites are the only naturally occurring extracellular stage of the erythrocytic cycle and since merozoites may be particularly immunogenic (Cohen 1979), we have here used highly purified merozoites from culture to induce immunity. We were especially interested in finding out whether immunity could be produced with merozoites from a line of P. falciparum that does not form the visible surface alterations of the infected erythrocyte called ‘knobs’ (Langreth et al. 1979). These knobs probably function as an attachment of cells bearing late-stage parasites to capillary endothelium and hence in the sequestration of these stages (Udeinya et al. 1981).

Materials and methods

P. falciparum cultures

The culture methods were essentially as described earlier (Trager & Jensen 1976, 1980). The medium was RPMI 1640 with HEPFS at 25 mM and type A human serum a t lo%, and the erythrocytes were type A+ human cells purchased from the New York Blood Centre. Two lines of FCR-3/Gambia (see Jensen & Trager 1978) were kept as stock cultures, each in two flow vessels (Trager 1979). The first was a wild-type, highly knobby line, designated F-86, that was selected every 4 weeks to favour the knobby trait. This selection was carried out in the following way. On a Friday the cultures were treated with 5% sorbitol (Lambros, & Vanderberg 1979) to leave mostly young stages. Three days later, when the parasites had multiplied through one 48 h cycle and late stages were prevalent, the cultures were treated with gelatin (Jensen 1978). Chiefly knobby late stages fail to sediment and stay in the supernatant (Langreth et al. 1979, Reese, Langreth & Trager 1979). These were then mixed with fresh red cells and allowed to reinvade for 4 h. The suspensions were then treated again with sorbitol to leave only the rings 0-4 h old. This procedure both selected for the knobby trait and synchronized the cultures. After one or two cycles in flow vessels, such cultures were inoculated to large flat-bottomed vessels (Trager & Jensen 1980).

The second line was a knobless (K-) clone (D-3) prepared by microscopic selection (Trager et al. 1981). This line was partially synchronized with sorbitol before being subcultured to iiirge vessels.

With each line, synchronization was repeated two cycles before merozoites were to be collected, and hence at the time of expansion of the cultues, usually to eight large vessels. The combined gelatin-sorbitol treatment was used for the F-86 material, sorbitol alone for the D-3. A single day’s preparation was usually made with material from eight large vessels, each holding 70 ml culture medium with 4 ml packed erythrocytes with a parasitaemia of about 10%. The preparation was begun when there appeared to be a maximal number of later schizonts.

Immunization with merozoires 221

Preparation of merozoites

This procedure has been described in detail (Stanley et al. 1982). Late-stage schizonts from synchronized cultures were suspended in medium with 10% human erythrocyte extract and incubated for 4 h at 37°C. The merozoites which emerged during this period were separated from most of the red cells and red cell membranes and other debris by differential centrifugation in 55% and 40% Percoll. The merozoites, which appeared dark from contamination with haemozoin, were washed finally with medium without serum and slides prepared for the determination of the number of merozoites and the extent of red cell contamination. Samples for electron microscopy were also prepared. We have shown previously (Stanley et al. 1982) that merozoites so prepared are in good condition morphologically and relatively little contaminated with red cells or red cell membranes. The final merozoite suspensions were stored in a dry-ice box at approximately -70°C.

Full information concerning each merozoite preparation used for immunization is given in Table I .

Monkeys; immunization; challenge

The nine Aotus trivirgatus monkeys selected for the experiment were received from South American Primates, Miami, Florida, in May 1980. They were housed in individual cages and were fed a special diet three times a week and softened monkey chow on the other days. The special diet was a thick liquid mixture that included bananas, eggs, orange juice, high protein baby cereal, sugar and multi-vitamins. All monkeys were of karyotype 2 (as determined by Dr Nancy Ma, New England Primate Center). Soon after arrival at the Rockefeller University they were vaccinated for both herpes simplex and herpes tamarinus. The vaccine was obtained from New England Primate Center. They were of similar weight (Table 2).

Thick blood films taken before and during immunization were negative for parasites. Blood for serum (3 ml) was drawn before and at intervals during and after immunization. For bleeding from a femoral vein, for intravenous inoculation or for intraperitoneal injection, the monkeys were first immobilized with an intramuscular injection of 8-10 mg ketamine HCI (Ketalar, Parke-Davis, 100 mg ketamine/ml). For intramuscular injection, for blood for thick and thin films and for haematocrits the monkeys were held by an assistant.

The emulsions for immunizations were prepared in the following way. The contents of a vial with 2 mg muramyl dipeptide (MDP) and 10 mg lactose, lyophilized (obtained through the kindness of Dr J.-P. Lacombe, Groupement d’Intt5ret Economique, Institut pour la Recherche et la Production d’Immunostimulants, Paris) were dissolved in 3 ml sterile saline (0.85% NaCI). One millilitre of this was used to suspend the F-86 merozoite preparations that were pooled for the first dose (see Table 1). Enough saline was added to the pooled material to make a total volume of 2.4 ml. This was then emulsified with 2.4 ml Freund’s incomplete adjuvant using two Luer-Lok syringes and a connecting 20 gauge needle. One-third of this emulsion was administered intramuscularly to each of Aotus monkeys 102, 108 and 110 (see Table 2). A second 1 ml portion of the MDP solution was used to prepare a similar emulsion of freshly thawed pooled merozoites of the clone D-3 for the first dose (see Table 1). This was injected in Aotus monkeys 11 1,112 and 1 13 (Table 2). The third 1 ml portion of MDP solution was used for thecontrol emulsion in which the

228 W. Trager et al.

Table 1. Merozoite collections used for injection of the monkeys

Contaminants per Date Total No. 10000 merozoites* Total No.

Parasite Dose Prep. of merozoites RBCt line No. No. prep. ( x lo9) RB P IRBC RBC ( x los)

F-86 1st

Total

2nd

Total

D-3 1st

Total

2nd

Total

1 1 Jun81 2 21 Jun81 3 5Jul81 4 8Jul81 5 8 Jul81 6 8Jul81 9 21 Jul81

7 14Jul81 8 14Jul81

10 21 Jul81 11 26 Jul81 12 29Jul81

3 23 May 81 4 13May81 8 6 June 81

1 19May 81 2 19May81 5 23 May 81 6 30May8l 7 3Jun81 9 6Jun81

1.1 0.7 0.6 0.4 0.3 1 -4 0.9 5.4

2.0 0.4 0.6 1 .o 1.1 5.1

3.6 1 * 5 0.4 5.5

1.2 0.9 1.6 0.6 1.2 0.3 5.8

920 5 3 1042 19 4 740 9 3 834 9 6 900 15 6 802 9 2

lo00 11 21

1351 17 1 1590 20 8 1250 8 34 970 29 7

1100 20 14

1300 16 23 1800 10 15 2211 19 10

850 14 37 lo00 10 22 2500 27 13 1063 14 15 I500 10 20 1650 11 11

1 1 0 0 0 0 2

0 0 2 7 2

0 1 0

11 3 0 0 2 0

4.4 3.5 1.8 1.2 1.8 2.8

20.7 36.2

2.0 3.2

21.6 14.0 17.6 58.4

82.8 24.0

5.2 112.0

57.6 22.5 20.9 9.0

26.4 3.3

139.6

* RB =residual bodies, P = extracellular parasites other than merozoites, IRBC =in- fected red cells, RBC =Uninfected red cells. t Calculated as follows:

( r R B ~ ~ ~ B C ) x total No. merozoites.

Thus for F-86, prep. 1, this would be:

%x 1.1 x 109=4.4x 10’.

Immunization with merozoites 229

Table 2. Immunization schedule (all monkeys were of karyotype 2)

Treatment

Aofus Weight 1st: 6 Oct 81 2nd: 27 Oct 81 No. Sex (g)* i.m. i.p.

106 M 1025 Each: 3 x lo6 RBCt Each: 4.6 x lo6 RBC 109 M 875 +220 pg MDPt f220pg MDP 116 F 950

102 M 925 Each: 1.8 x lo9 MZt . Each: 1.7 x lo9 MZ

110 F 860 +22Opg MDP +220 p g MDP 108 M 850 OfF-86 of F-86

1 I I M 825 ~ a c h : 1.8 x 109 MZ ~ a c h : 1.9 x 109 MZ 112 M 1100 ofD-3clone of D-3 clone 113 M 800 +220pgMDP + 220 pg MDP

* On 9 October 1981. t RBC = red blood cells, MZ = merozoites. See Table 1 for details of preparations.

MDP: 2 mg MDP+ 10 mg lactose in 3 ml saline. 1 rnl or 670 pg MDP, used for each emulsion for three monkeys so that each monkey received 220 pg MDP. There were no visible reactions to any of the treatments. All monkeys were challenged with 4.5 x lo' prasites intravenously on 25 November 1981.

appropriate number of normal human type A+ erythrocytes, previously washed and stored frozen at - 70°C, was suspended. This was injected into monkeys 106,109 and 1 16. The intramuscular injections of this first dose were all given in the right thigh with a 22 gauge needle.

The emulsions for the second dose were similarly prepared (Table l) , but they were administered intraperitoneally (Table 2). Each monkey was immobilized with ketamine. A blood sample was taken from the femoral vein and then the emulsion was injected intraperitoneally to the right of the midline using a 23 gauge needle. There was no leakage from the sites of injection and the monkeys did not develop any visible tissue reaction a t the injection site.

Parasites for the challenge inoculation were prepared as follows. One Aotus of karyotype 3 had been infected with material from the culture line F-86 that was cultured for 4 days in Aozus erythrocytes. This monkey developed a parasitaemia of 20%, at which time its blood was frozen and stored in liquid nitrogen. On 16 November 1981, a vial of this blood was thawed and inoculated intravenously to Aotus 105 (karyotye 3). On 25 November a thin film from this monkey showed six rings per 10000 red cells. At this time, 3 ml blood were taken into heparin and 0.15 ml of this whole heparinized blood was

230 W . Trager et al.

inoculated intravenously to each of the nine experimental monkeys. Since this blood had a red cell count of 5 x lo6 per mm3, each monkey received 4.5 x los parasites. The order of inoculation was 102, 111, 106, 108, 112, 109, 110, 113, 116.

Daily thin films were begun on 1 December, the 6th day after challenge, and continued through 18 December (except for 6 December). The one monkey showing a severe infection (1 16) received 1 ml quinine hydrochloride (50 mg/ml solution) intramuscularly on 17 December; all the others on 22 December, by which time all parasitaemias were reduced.

Indirect fluorescent antibody test (IFA)

The methods of Collins & Skinner (1972) were followed with slight modifications. All the monkey sera were inactivated at 56°C for 30 rnin and then adsorbed with normal human type A+ erythrocytes. For each adsorption, 1 ml serum was mixed with 0.5 ml pooled cells and held at 4°C for $ h for the early treatments and overnight for the next to last treatments. Adsorptions were repeated until no agglutination could be seen and then once more.

The antigen-bearing cells were prepared by gelatin flotation from a stock culture of a knobby clone (clone I) of FCR-3/Gambia. This gave slides with a high proportion of large parasites. The unfixed blood films were stored frozen at - 20°C. They were dehaemoglo- binized by 5 min in 0.1% HCI, washed for 5 min in distilled water and twice in phosphate-buffered saline (PBS). Dilutions of the test sera were then applied and incubated for 30 min at 37°C. After three washes in PBS, fluorescein isothiocyanate- labelled anti-human IgG, IgA and IgM (Miles-Yeda), diluted 1 : 20 in PBS, was applied and the slides again incubated for 30 min at 37°C. The slides were now washed three times in PBS, mounted in nine parts glycerin with one part PBS and examined with a Zeiss epifluorescence microscope. The highest dilution of antiserum giving a distinct fluores- cence was considered the end-point.

Assessment of condition of merozoite preparations

The extent of contamination with intact red cells was determined by counts on stained slides (Table 1). The morphological condition of the merozoites and the amount of extraneous membranes and other contaminants were assessed from electron micrographs (see Figures 2 & 3). Samples for electron microscopy were fixed for 1 h at room temperature in 2% (v/v) glutaraldehyde with 0.12 M sucrose, 0.2 PM CaC12, buffered with 0.1 M Na cacodylate-HCl at pH 7.4. After postfixation in 1.5% (w/v) OsO4 with 0.12 M sucrose and 0.1 M cacodylate-HC1 buffer, en bloc staining with uranyl acetate, samples were dehydrated and embedded in Epon 812 (Langreth et al. 1978). Uranyl-acetate- and lead-citrate-stained sections were examined with a Philips 300 electron microscope.

Results

The principal results of this immunization experiment are shown in Figure 1 and Table 3. The three control monkeys had prolonged infections with fluctuating parasitaemias, frequently over 20 parasites per 10000 red cells and reaching peaks of 40, 60 and 220

Immunization with merozoites 23 1

Table 3. Infections after challenge on 25 November 81

Days after inoculation to: No. of days with

First parasitaemia Parasites per parasitaemia of 20/10000 Aotus at 10/10000 RBC Peak 10000 RBC RBC or higher during 23 No. or higher parasitaemia at peak day course of infection

106 109 116

I02 108 110

1 1 1 112 113

7 10 8

t 12 13

13 13 13

12 13

(13)* 21

13 13

13 16 13

41 10 60 5

(67)* 221 10

106 14

12 22 34

9 0

0 1 1

* Two peaks; first on day 13, followed by a decrease, then a rise to day 21, then a decrease (after quinine on day 22). t This monkey died with intravascular haemolysis on day 12, parasitaemia 8/10000 RBC.

t 34

6 8 10 12 14 16 18 20 22 Days after challenge

Figure 1. Daily parasitaemia as parasites per 10000 erythrocytes during the period from 1 December 1981 to 18 December 1981 for the control group (A=monkeys 106, 109 and 116), for the group vaccinated with merozoites of the K- clone D-3 (B = monkeys 11 1,112 and 113) and for the group vaccinated with merozoites of the K+ strain (C=monkeys 102,108 and 110). No slides were taken on 6 December. (See also Tables 1 and 2.) Monkey 116 received quinine on 17 December, the others on 22 December (27th day after challenge). On 29 December, monkeys 106, 109,116, 108, 110 and 1 I 1 were positive in thick films but no parasites could be found in monkeys 112 and 113. By 14 January 1982, parasites were found in thick films only in monkeys 106, 116 and 108. No recrudescence was observed.

232 W.Trager et al.

Figure 2. Low magnification electron micrograph of a merozoite preparation (F-86, No. 2) obtained from the Kf line. The preparation consists mainly of merozoites, residual bodies, and membranous elements. Bar indicates 1 pm. x 6400. Figure 3. Higher magnification electron micrograph of a K - merozoite preparation (D-3, No. 7). Note the well-preserved structure and the brushy surface coats forming contacts between neighbouring merozoites (arrow). Bar indicates 0.5 pm. x 37500.

parasites per 10000 red cells. The three monkeys that had received the merozoites of the K- clone D-3 had very low parasitaemias, well under 20 per 10000 red cells, except a t one peak when one reached 22 and another 34. Of the three monkeys that had received merozoites of the K+ line F-86, one died early with an intravascular haemolysis and a parasitaemia of only eight per 10000 red cells. Of the other two, one had an infection like that of the controls whereas the other had a very low-grade infection.

The IFA reaction was negative for all monkeys before the start of the experiment. After the first immunizing dose, the monkeys that had received merozoites had positive IFA titres (Table 4), with the exception of the one that later died with intravascular haemolysis. Even after the second immunizing dose, this monkey still had a low titre of only I : 160 as compared with 1 : 1280 or higher for the other five vaccinated monkeys. The controls were negative (one with a titre of 1 : 20) before challenge. After challenge all the monkeys had positive IFAs with appreciably higher titres for the immunized ones than for the controls.

The haematocrit was decreased in all monkeys during the course of infection (Table 5) . This was most marked in two of the three control animals, falling to only 11% in the

Immunization with merozoites 233

Table 4. IFA titres of serum samples obtained during immunization and after challenge. The monkeys were injected on 6 October and 27 October 1981 (see Table 2) and challenged on 25 November 1981 (see Table 3). All sera were negative before start of immunization

Highest dilution of serum giving distinct fluorescence Aotus No. Sample of 27 Oct 81 Sample of 10 Nov 81 Sample of 22 Dec 81 t

106 109 I16

Neg. at 1 : lo* 1 : l O

Neg. at 1 :20

Neg. at 1 :20* 1:20

Neg. at 1 : 20

1:160 1 :640 1:160

I02 108 110

Neg. at 1 : 10 1:80 1 :40

1:160 1 : 2560 1 : 2560

- 1 : 5120 1 : 10240

1 1 1 I12 1 I3

1 :80 1:20 1:80

I : I280 1:5120 1 : 2560

1 : 1280 1:5120 1 : 10240

* Lowest dilution tested. t After challenge.

monkey with the highest parasitaemia. All were back at or close to normal 3 weeks after treatment with quinine. No other pathological effects of either the immunization procedure or the infection were seen, except for the single individual that died.

Table 5. Haematocrits (%) of monkeys after immunization and during and after infection. The monkeys were challenged on 25 November 1981

Aotus No. 10 Nov 81 1 Dec 81 4 Dec 81 10 Dec 81 15 Dec 81 22 Dec 81 14 Jan 82

106 46 I09 45 116 38

52 47 47

50 38 48

35 25 27 26 15 27 37 11 -

47 43 52

I02 39 108 48 110 46

46 49 53

25* 51 40

32 45 38 33 41

- 52 52

1 1 1 43 112 47 1 I3 41

31 33 44

29 34 37

31 20 22 29 28 I9 30 46 43

42 34 50

* Died with intravascular haemolysis on 7 December 1981.

234 W. Trager et al.

Discussion

The most important conclusion to be drawn from the results here presented is that purified merozoites derived from a knobless clone of a strain of parasites long maintained in culture can serve as antigens to confer protective immunity against the wild-type knobby parasites of the same strain. It is worth some emphasis that, unlike previous immunization experiments, the controls received not adjuvant alone but adjuvant with that amount of red cells estimated as the maximal contamination of the merozoite preparations given to the vaccinated animals. This contamination was not large. Besides the small number of intact red cells, other contaminants of the merozoite preparations were residual bodies, already noted to have surface coats like those of merozoites, and membrane vesicles, which did not react with either anti-erythrocyte or anti-parasite antibodies (Stanley et al. 1982). It has already been observed that K + and K- merozoite surfaces react in the same way in a ferritin-labelled antibody assay using serum from P. fakiparum-immune Aotus (Stanley et al. 1982). We have now shown that knobless merozoites from a clone protect against challenge with wild-type parasites. This protection suggests that the knob material, shown to be antigenically reactive (Kilejian, Abati & Trager 1977, Langreth & Reese 1979), may not be essential for the production of some protective immunity.

The results unfortunately cannot tell us whether purified merozoites derived from the wild-type knobby strain are as effective as those from the knobless clone. In the group of three monkeys that received wild-type merozoites, one died early with a low parasitaemia and intravascular haemolysis. Autopsy of this monkey revealed two major lesions that contributed to its death: (1) cardiac failure with pulmonary congestion and fibrosis; (2) hepatic necrosis. Since the parasitaemia at death was very low, the haemolysis and death may have been only indirectly related to the malarial infection. The exceptionally low IFA response of this monkey also suggests some immunological abnormality. If the remaining two monkeys of this group are considered together with the three that received merozoites of the knobless clone, we have a group of five vaccinated animals. Of these, four were highly protected whereas one showed a parasitaemia like that of the controls (Table 3).

The relatively low levels of parasitaemia in the controls are assumed to result from the use for challenge of parasites that had previously been passed only twice through Aotus. The possibility cannot be excluded that they were also a result of a non-specific immunostimulatory effect of MDP. Because of the limited supply of Aotus we considered it more important to have controls that received red cells and MDP rather than untouched controls.

A second conclusion of some importance to be drawn from these experiments is that MDP can serve as an effective adjuvant, a conclusion that is not surprising in view of earlier results (Reese et al. 1978, Siddiqui et al. 1978,1979). Quite apart from discussion as to the ultimate utility of MDP or its analogues (Chedid et al. 1976, Lederer 1980) in a vaccine for human use, the efficacy of MDP in work with Aotus provides a material far less traumatic to the experimental animals than Freund’s complete adjuvant. Accordingly it should prove valuable in the extensive testing yet to be done both with whole organism antigens of the kind used here and with selected parasite proteins thought to be involved in induction of protective immunity (Brown et al. 1981, Perrin, Dayal & Reider 1981, Reese, Motyl & Hofer-Warbinek 1981).

Immunization with merozoites 235

Acknowledgements

It is a pleasure to acknowledge the outstanding assistance of Mrs Marika A. Tershakovec, Mrs Liubov Lyandvert, Mr Erminio Gubert and Mr James Stanorski. The work was supported by a Contract (DSPE-C-0030) from the United States Agency for Interna- tional Development and by a grant from the UNDP/World Bank Special Programme in Tropical Diseases of the World Health Organization. We are grateful to Dr E. Lederer and Dr J.-P. Lacombe for gifts of muramyl dipeptide.

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