Immunology, 1975, 29, 397.

Merozoite Vaccination Against Plasmodium knowlesi Malaria G. H. MITCHELL, G. A. BUTCHER AND S. COHEN Department of Chemical Pathology, Guy's Hospital Medical School, London

(Received 10th December 1974; accepted for publication 28th January 1975) Summary. Free malarial merozoites isolated from in vitro cultures of P. knowlesi and emulsified with Freund's complete (FCA) or incomplete (FIA) adjuvant were used to vaccinate twelve Rhesus monkeys against the uniformly lethal infection caused by P. knowlesi. Initial challenge of six monkeys with the same parasite variant as used for vaccination produced no detectable infection in three monkeys, while three others developed low-grade parasitaemia (maximum 1b5 per cent), which terminated after 6-11 days. Vaccination with merozoites in either FCA or FIA induced protection against homologous variant challenge. Six other monkeys were challenged first with a parasite variant different from that used for vaccination. Two animals immunized with merozoites in FIA alone or in FCA on only one occasion developed fatal infections. The other four animals vaccinated at least twice with merozoites in FCA showed low-grade parasitaemia (maximum 15 per cent) which terminated after 8-12 days. Eight monkeys rechallenged on eleven occasions at intervals of up to 16 weeks were completely resistant to several variants and a distinct laboratory strain of P. knowlesi, but developed chronic malaria similar to that in unimmunized controls when challenged with a different species of malaria, P. cynomolgi bastianellii. It is concluded that merozoite vaccination of Rhesus monkeys induces immunity against the erythrocyte stages of P. knowlesi far greater in degree and significantly broader in variant specificity than that achieved by previous methods of immunization or by repeated drug-controlled infections.

INTRODUCTION Plasmodium knowlesi malaria is uniformly fatal in Rhesus monkeys (Garnham, 1966). If infections are initially controlled with drug therapy, however, a degree of immunity can be induced associated with periodic relapse and low grade infection after repeated challenge. In such resistant monkeys each recurring infection is attributable to a distinct parasite variant recognized by the schizont-infected cell agglutination (SICA) test (Brown and Brown, 1965). Antigenic variation therefore seems to be an important mechanism determining chronicity of infection (Brown, 1971) and complicates any attempt to achieve effective immunoprophylaxis (Cohen, 1974). Rhesus monkeys have been vaccinated with formol-treated (Freund, Thomson, Sommer, Walter and Pisani, 1948; Targett and Fulton, 1965) or freeze-thawed (Brown, Brown and Hills, 1970) erythrocytic schizonts of P. Correspondence: Professor S. Cohen, Department of Chemical Pathology, Guy's Hospital Medical School, London SEl 9RT.


G. H. Mitchell, G. A. Butcher and S. Cohen 398 knowlesi or fractions of these parasites (Simpson, Schenkel and Silverman, 1974; Schenkel, Simpson and Silverman, 1973) in complete (FCA) or incomplete (FIA) Freund's adjuvant. Vaccination reduced mortality to about 50 per cent after challenge with the immunizing variant, but provided no protection against challenge with other variants of the same strain of P. knowlesi (Brown, Brown and Hills, 1970). The protection achieved always required the use of complete adjuvant, but this alone was inactive. Studies on the mechanism of acquired malarial immunity in vivo (Cohen, McGregor and Carrington, 1961) and in vitro (Cohen, Butcher and Crandall, 1969) indicated that protective antibody did not affect intracellular parasites, but interrupted the cycle of development at the stage when merozoites were released into the plasma. In addition, immune serum agglutinated free merozoites (Butcher and Cohen, 1970) and prevented their attachment to receptors which were demonstrated on the red cells of susceptible species (Butcher, Mitchell and Cohen, 1973). These findings all indicated an important role for merozoite antigens in specific malarial immunity and prompted development of an in vitro method for isolating extracellular blood stage merozoites (Mitchell, Butcher and Cohen, 1973). Here we describe the use of these preparations for vaccination of monkeys against P. knowlesi malaria. Part of this work has been the subject of a preliminary report (Mitchell, Butcher and Cohen, 1974)

MATERIALS AND METHODS Animals Imported Rhesus monkeys (Macaca mulatta) and Kra monkeys (M. fascicularis) of either sex weighing 2-4 kg were caged in a room artificially illuminated between 7.00 a.m. and 7.00 p.m. Parasites P. knowlesi parasites were originally obtained from: (i) a strain maintained at the Walter Reed Army Institute of Research Washington and designated W (Butcher and Cohen, 1972); (ii) a strain maintained at the National Institute for Medical Research, London, and designated Nuri (Brown, Brown and Hills, 1968). P. cynomolgi bastianellii was provided by Dr D. E. Bidwell of the Nuffield Institute of Comparative Medicine.

Identification ofparasite variants

Serologically distinct variants of the W and Nuri strains of P. knowlesi were identified by the schizont-infected cell agglutination (SICA) test (Brown et al., 1968). These variants maintained their specificity unchanged after storage at -80° in 15 per cent glycerol-saline or during repeated passage through normal Rhesus monkeys (Butcher and Cohen, 1972). Merozoite preparations Merozoites were isolated by centrifugation from in vitro cultures of P. knowlesi (W1 variant) using the method described by Mitchell, Butcher and Cohen (1973) except that normal and parasitized red cells were agglutinated by phytohaemagglutinin (1.5 mg/ml of cultured cell suspension) and not by antiserum. The yield of merozoites was approximately 2 x IO" per millilitre of cultured erythrocytic schizonts and contamination with normal or parasitized red cells was less than 0 1 per cent.

Vaccination against Malaria


Vaccination Freshly isolated merozoites suspended in 1 ml of medium 199 (Burroughs Wellcome) were emulsified with equal volumes of Freund's complete (FCA) or incomplete (FIA) adjuvant. This material was administered within 2 hours of merozoite isolation by intramuscular injection in doses, and according to schedules, shown in Tables 1 and 3. In control experiments, mature segmented schizonts freshly isolated from in vitro cultures and emulsified with FCA were injected as shown in Tables 1 and 2. Challenge of immunized animals Immunized monkeys were challenged by intravenous injection of 104 or 105 immature schizonts of known strain and variant specificity taken from an infected donor. Equivalent challenge infections were administered simultaneously to unimmunized control animals (Tables 1 and 4).

Parasite counts The first appearance of parasitaemia was assessed on thick blood films haemolysed and stained in Field's solution. On the basis of the number of leucocytes observed, a negative blood film was thought to indicate a parasite density of less than 1 in 106 red cells. Parasite numbers were estimated by erythrocyte counts and the percentage of red cells parasitized was determined by counting up to 104 erythrocytes on thin blood films. Blood was examined daily for at least 14 days after each immunization and five or more times weekly from the time of challenge and until blood films had been negative for at least 21 days.

Test for sub-patent infection The possibility that monkeys showing negative blood films might harbour a sub-patent infection was examined in some instances by the intravenous injection of 5 ml of blood into a normal Rhesus. Blood films from the recipient were examined as described above. RESULTS VACCINATION OF M. fascicularis Our initial experiments were performed on Kra monkeys, which are the natural hosts of P. knowlesi (Garnham, 1966). All had been captured from the wild state in South East Asia and had presumably been exposed to P. knowlesi malaria; the animals had been maintained in a laboratory in the U.K. for at least 6 months. Before the present experiments, blood smears from these monkeys were consistently negative and their sera did not inhibit in vitro multiplication of P. knowlesi (Cohen et al., 1969). One monkey was immunized with P. knowlesi mature schizonts (WI) in FCA and another with merozoites (WI) in FCA as shown in Table 1. The control and schizont-immunized animals challenged with P. knowlesi became infected on each occasion; parasitaemia lasted for up to 10 days and did not exceed 0*6 per cent (Table 1). The merozoite-immunized animal challenged five times over a period of 146 days, remained negative on four occasions and showed parasitaemia (less than one parasite per 105 red cells) on only 1 day after the fifth challenge with the N strain of P. knowlesi (Table 1). These findings encouraged a more comprehensive investigation of merozoite immunization in Rhesus monkeys in which P. knowlesi causes a rapidly fatal infection.


G. H. Mitchell, G. A. Butcher and S. Cohen TABLE 1 IMMUNIZATION


M. fascicularis

P. knowlesi MALARIA


Challenge (104 parasites)


Monkey Preparation

Number* (day)



Mature schizonts



1 x 108, 2 x 107, 1 x 107 (52) (1) (16) 5 x 108, 2 x 109, 1 x 109 (75) (82) (1)



Days patent§

1 67 86 147 177 98 126 196 221 244


5-13 (46) 72-75 (92) 95-109 153-158 (158) 184-194


Maximum number of parasites per 104 red blood cells 50 3 60

01 11 Negative Negative Negative

Negative 0.1


* Number of parasites in Freund's complete adjuvant.

t This refers to the time after first challenge (control) or after first inoculations (vaccinated). t W = Walter Reed strain and N = Nuri (Mill-Hill) strain of P. knowlesi. Numbers denote distinct variants

differentiated by the SICA test (see text). § Number in parentheses shows day of drug cure.


Monkey Szl Sz3

Challenge (104 parasites) Vaccination* Number of schizonts (day) Dayt Parasitet Days patent§ Maximum Subinoculation (day) * * parasitaemia 1 x 108, 2 x107, 1 x 107 (52) (1) (16) 1 x 108,2x 107, 1 x 107 (1) (41) (77)

86 147 170 91




Negative 175-186 94-100 D

I (161)

8 2500

* Number of schizonts in Freund's complete adjuvant. to the time after the first challenge (control) or after the first inoculation (vaccinated). I§ W == Walter Reed strain and N = Nuri (Mill-Hill) strain of P. knowlesi. D died on day shown. ** Subinoculation of 5 ml of blood from vaccinated into normal Rhesus on day shown. I = infection

t This refers in recipient.

M. mulatta Two Rhesus monkeys were immunized with mature schizonts of P. knowlesi (Wi) and twelve with merozoites (WI) in FIA or FCA according to the doses and schedules shown in Tables 2 and 3. Immunization never produced detectable infection. Animals were challenged with parasites of known strain and variant specificity on days shown in Tables 2 and 3. Equivalent challenge infections were administered simultaneously to unimmunized Rhesus and all suffered progressive infections. Nine out of twelve of the control animals died within 8-11 days and three others were cured on day 5 or 6 when the level of parasitaemia indicated a fatal outcome (Table 4). VACCINATION OF


against Malaria






Number of parasites in FCA (day)


Challenge (104 parasites)




Number of parasites in FIA (day)

Day* Parasitet Days patent

Maximum Subinocunumber of parasites per 104 red



S (82)

blood cells

Mz2O MzI8 Mz22

Mzl2 MzlO

5x 109 (1) 2-3 x 109 (1)

1-6 x 109 (36) 5x108 2x109 1x109

Mz2l Mzl9¶ Mz23

Mz14 Mz15



85 51 162 63 85 98


(75) (82) 5x109 6x109 5x10)

74 93 127 232 280 6x 108 9x 108 2x 109 5x 109 113 148 (1) (14) (62) (98) 2 3x 109 1 6x l09 71 (1)


2-3 x 108 1-6 x 109 (1) (36) 2 x 109 (17)



(1) 2 x 109 (17)

5 x 109

(1) 23x109 16x109 (1) (36) 6 x 108 9 x 10(8 2 x 109 (1) (14) (62) 6 x 108 9 x 108 2 x 109 (1) (14) (62) 6x 108 9x 108 2x 109 5x 109 (1)





WI W3 Wl W3 Wl W3 WI WI** W42 W12 WI Ni WI W3 W3




Negative 22 Negative


Negative Negative

S (85)



S (133) I (259)


Negative 4 Negative

I (124)

Negative Negative








76 118 76 118 112 148

W3 W42 W3 W42 W3




D 75








Negative 28 Negative

* This refers to the time after first challenge (control) or after first inoculation (vaccinated). = Walter Reed strain and N = Nuri (Mill-Hill) strain of P. knowlesi. Numbers denote distinct variants differentiated by the SICA test (see text). *§ D = died on day shown. Subinoculation of 5 ml of blood from normal Rhesus on day shown. I = infection in recipient. S = sterile on subinoculation. ¶ See text for pathological complications in this animal. ** Challenge with 10f P. knowlesi parasites.




Six merozoite-immunized monkeys were challenged 2-5 weeks after the last inoculation with the same variant (WI) as used for immunization. No infection was detected in three of these animals (Mz 18, 12 and 10, Table 3) and subinoculation of 5 ml of blood from one (Mz 0) on the 11th day after challenge revealed no viable parasites. Three other merozoiteimmunized animals challenged first with the WI variant (Mz2O, 22 and 16, Table 3) showed parasitaemia after 5-18 days; parasites remained detectable for 6-8 days and reached peak levels of 0-04-1 5 per cent in the individual monkeys. The various schedules of merozoite immunization (Table 3) appeared to induce comparable immunity. The monkey (Mz2O, Table 3) immunized with merozoites in FIA showed no delay in patency (Fig. 1), and the infection, which reached a maximum of 1-5 per cent, lasted for 8 days



Parasite (variant)*

Patent (day)

Maximum parasitaemia (per 104 red blood cells)t

Death (day)

WI WI WI WI W3 W3 W3 W3 W8 W12 W42 Ni

3 3 3 5 4 3 4 4 3

1800 670 320 1500

8 11

3000 1900 1100 700 3000



3 4



Drug cure (day)


11 9 7

9 9 8 6 6

* W WValter Reed strain and N Nuri (Mill-Hill) strain of P. knowlesi. Numbers denote distinct variants differentiated by the SICA test (see text). t Parasitaemia on the day before death or drug cure. =


and was then completely cleared as judged by a negative subinoculation test on the 19th day after challenge. One monkey vaccinated three times with mature schizonts in FCA (Table 2) and challenged after 5 weeks with the immunizing variant (WI) developed parasitaemia after 9 days, which persisted for 11 days with a maximum of 1 3 per cent (compare Brown et al., 1970). INITIAL CHALLENGE WITH A HETEROLOGOUS VARIANT (W3)

Six merozoite-immunized animals were first challenged 2-5 weeks after the last immuization, with a P. knowlesi variant (W3) different from that used for vaccination. One monkey (Mz2l, Table 3) inoculated with merozoites in FIA showed no delay in patency, developed progressive parasitaemia and died 2 days after the control animal (Fig. 1). Another animal (Mz19, Table 3) immunized first with merozoites in FCA and then in FIA, showed a 2-day delay in patency, became clinically ill when parasitaemia was only 1 per cent and died 16 days after challenge when parasitaemia was 7 per cent. On the day of death the erythrocytic count was 2 x 106/mm3 and the blood urea above 300 mg/100 ml. Post-mortem examination revealed a chronic pleural abscess infiltrating the diaphragm and upper portion of the liver. The cause of death was not obvious, but parasitaemia had reached a potentially fatal level (25 per cent) 3 days before death. The four remaining monkeys received two or more inoculations of merozoites in FCA. All developed patent infections 5-7 days after W3-variant challenge; parasitaemia persisted for 1-12 days and reached maximum levels of 0 1-1I5 per cent in the individual animals (Fig. 2). One Rhesus immunized three times with mature schizonts in FCA and challenged with the W3 variant developed a fatal infection indistinguishable from that in control animals (Table 2, Fig. 2) (compare Brown et al., 1970).


Vaccination against Malaria 40r 30 0

._E £



Ca CL v a C 0,








I0 I5 20 0 Days after challenge



FIG. 1. Course of parasitaemia in (a) three monkeys challenged first with the immunizing variant (W1) of P. knowlesi and (b) another three monkeys challenged first with a different variant (W3). Immunization with merozoites in FCA protected against initial challenge with both variants; immunization in FIA protected against the immunizing variant, but not against first challenge with a different variant (see Table 3). (@) Control, died day 11. (-) Mz2O FIA (1-5 per cent). (v) Mz22 FCA (0-22 per cent). (0) Control, died day 9. (o) Mz2l (FIA), died day 1 1. (0) Mz23 FCA (0-001 per cent). Figures in parentheses indicate peak parasitaemia. Level of parasitaemia is shown on a logarithmic scale between 0 001 and 1 0 per cent.















Dnrs after challenge

FIG. 2. Course of parasitaemia in two normal Rhesus monkeys, one immunized with schizonts (Sz3) and four immunized with merozoites (MzI4, 15, 17 and 23) in FCA on two or more occasions. All animals were immunized with WI variant and challenged first with W3 variant parasites. To illustrate the course of low-grade infections in the merozoite-immunized animals, parasitaemia is shown on a logarithmic scale between 0-001 and 1-0 per cent. (0) Control, died day 7. (o) Control, died day 9. (0) Sz3, died day 9. (a) Mz14 (0-07 per cent). (A) Mz15 (15 per cent). (*) Mz23 (< 0-001 per cent). (v) Mz17 (0-28 per cent). Figures in parentheses indicate peak parasitaemia.


G. H. Mitchell, G. A. Butcher and S. Cohen SUBSEQUENT CHALLENGE OF IMMUNIZED


Eight animals were challenged a second time, usually with variants different from that used for immunization, at 5-16 weeks after the first challenge (Table 3). None of these monkeys developed a patent infection in response to the second challenge. Of particular interest is the monkey (Mz2O, Table 3) which was immunized with merozoites in FIA and challenged first with WI; this animal was completely resistant to subsequent challenge with the W3 variant. One monkey (Mz 10, Table 3) remained negative after a third challenge with W12 and was shown by subinoculation 16 days later to have a sterile immunity. This animal did eventually develop parasitaemia for 9 days (maximum 0-02 per cent) after a fourth challenge with WI given 33 weeks after initial immunization. Seven weeks after this Mz 10 was challenged with the Nuri strain of P. knowlesi and remained negative. A monkey immunized with schizonts in FCA was challenged for a second time with the immunizing variant and remained negative, but had a subpatent infection revealed by subinoculation (Table 2). Three weeks later, challenge with the Nuri strain produced a patent infection after 5 days which lasted for 11 days (maximum 0-08 per cent). SPECIFICITY OF IMMUNE PROTECTION

Two normal Rhesus, one immunized with schizonts and three immunized with merozoites, were challenged with 105 P. cynomolgi bastianellii parasites (Table 5). Control TABLE 5 P. cynomolgi bastianellii CHALLENGE OF NORMAL AND VACCINATED RHESUS MONKEYS

Monkey Control Control Szlt Mzl0

Mzl4j Mzl5t

Day of challenge* 213 323 142 142

Pre-patent Maximum parasitaemia Duration patent period (days) per 104 red blood cells infection (days) 5 4 5

12 4 5

1200 1250 650 68 400 550

> 80 > 80 > 80 > 80 > 80 > 80

* Day after initial vaccination on which P. cynomolgi bastianellii challenge was administered. t See Table 2 for previous vaccination and challenge. I See Table 3 for previous vaccination and challenge.

animals developed peak parasitaemia of about 12 per cent followed by chronic, low-grade infection. All immunized animals showed similar long-lasting infections, but maximum parasitaemia was lower than in non-vaccinated monkeys.

DISCUSSION Studies on the mechanism of malarial immunity (Cohen et al., 1961, 1969) prompted the idea that extra-cellular merozoites might provide the basis for a malarial vaccine. The elaboration of a simple method for isolating merozoites (Mitchell et al., 1973) allowed us to test this hypothesis. Initial experiments involving merozoite vaccination of M. fascicularis, the natural host of P. knowlesi (Table 1) gave encouraging results. The significance

405 Vaccination against Malaria of these data was, however, in doubt because the animals had almost certainly been exposed to malaria in the wild state and P. knowlesi produces chronic, non-lethal infections in this species. A more detailed study of merozoite vaccination was therefore undertaken in M. mulatta. P. knowlesi malaria is invariably fatal in the Rhesus monkey (Table 4) and this host-parasite combination therefore provided the most stringent of all models available for testing the effectiveness of a malaria vaccine. Several previous attempts have been made to immunize Rhesus monkeys against P. knowlesi malaria using killed erythrocytic parasites or fractions derived from these. In the majority of studies the parasites used for vaccination and challenge were said to be the same, but their serological identity was not established. For this reason the significance of results obtained by Freund et al. (1948), Targett and Fulton (1965), Schenkel et al. (1973) and Simpson et al. (1974) cannot be accurately assessed, but all these studies showed increased survival rates in animals vaccinated with malarial antigens in FCA. Brown et al. (1970) in a carefully controlled study vaccinated Rhesus monkeys with freeze-thawed erythrocytic schizonts of known variant specificity and subsequently challenged the animals with the same or a different variant of P. knowlesi. Seven out of thirteen monkeys given the schizont vaccine in FCA died following challenge with the variant used for immunization. The six animals which survived all developed parasitaemia which, in different individuals, reached maximum levels of about 3-10 per cent and lasted 8-15 days; subinoculation of blood from these monkeys into normal recipients showed that the parasites had been completely eliminated. Vaccines containing FIA provided no protection against homologous variant challenge. Three monkeys vaccinated with schizonts in FCA were challenged with a variant different from that used for immunization and all suffered fatal infections. Comparable results were obtained in our study of two animals immunized on three occasions with schizonts in FCA; homologous variant challenge produced transient parasitaemia, but challenge with a heterologous variant was fatal

(Table 2). In contrast to what was observed after immunization with parasitized cells or after repeated infection initially controlled with drug therapy (Voller and Rossan, 1969; Brown et al., 1970; Butcher and Cohen, 1972), vaccination with merozoites derived from a single serological variant of P. knowlesi was characterized by the following. (i) Uniform survival after initial challenge with the immunizing variant; such challenge produced no detectable parasitaemia or a transient infection (1-11 days) of low intensity (maximum parasitaemia 1-5 per cent); resistance to homologous challenge was observed after vaccination with merozoites in either FIA or FCA. (ii) Animals inoculated at least twice with merozoites in FCA uniformly survived initial challenge with a variant of P. knowlesi different from that used for immunization. All such monkeys developed patent infections, but these were of brief duration (1-12 days) and low intensity (maximum 1 f5 per cent). (iii) After initial challenge, all surviving immunized animals were strongly resistant to subsequent challenge made at intervals of up to 16 weeks with several variants of the W strain and a distinct strain of P. knowlesi. These monkeys remain susceptible to chronic infection by P. cynomolgi bastianellii. The results (Table 3) indicated that merozoite vaccination using FCA was required to induce immunity to initial challenge with a heterologous variant but that complete adjuvant was not essential for resistance to initial homologous variant challenge. However, the monkey (Mz2O, Table 3) vaccinated with incomplete adjuvant and challenged with

406 G. H. Mitchell, G. A. Butcher and S. Cohen the immunizing variant was subsequently completely resistant to heterologous variant challenge. This suggests that the method of vaccination could be modified to remove the requirement for complete adjuvant in the induction of immunity to a heterologous variant. We are at present investigating this possibility. The number of merozoites used for vaccination was chosen arbitrarily; a smaller number might be effective, but at the level used, the in vitro cultures provided about 20 immunizing doses per millilitre of parasitized red cells. Merozoite vaccination induced specific immunity far greater in degree and significantly broader in specificity than that previously achieved by immunization or repeated infection. This form of vaccination apparently stimulated a response to antigens present on a wide spectrum of P. knowlesi parasites, but poorly immunogenic during natural malaria infection. The response must also differ qualitatively from that observed after repeated drug-controlled infection, since the latter is associated with chronic lowgrade parasitaemia, whereas patent infections occurring in immunized monkeys never persist beyond 12 days. We are attempting to define the specific immune effector mechanisms induced by merozoite vaccination. The results reported here are significant with regard to the development of an effective vaccine against human malaria in view of the probable existence of immunologically distinct strains of P. falciparum (Sadun, Hickman, Wellde, Moon and Udeozo, 1966; Diggs, Wellde, Anderson, Weber and Rodriguez, 1972) and the established genetic diversity of this parasite in West Africa (Carter and McGregor, 1973).

ACKNOWLEDGMENTS This work was supported by the Medical Research Council, London, and the World Health Organization. We thank Miss Karen Smyth and Mr E. D. Dennis for their skilled technical assistance. P. cynomolgi bastianellii was supplied by Dr D. E. Bidwell of the Nuffield Institute of Comparative Medicine. REFERENCES

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infection.' Nature (Lond.), 244, 40. CARTER, R. and MCGREGOR, I. A. (1973). 'Enzyme variation in Plasmodium falciparum in the Gambia.' Trans. roy. Soc. trap. Med. Hyg., 67, 830. COHEN, S. (1974). 'Immunoprophylaxis of protozoal diseases.' Clinical Aspects of Immunology, 3rd edn (Ed. by R. G. H. Gell, R. R. A. Coombs and P. J. Lachmann, p. 1649. Blackwell Scientific Publications, Oxford. COHEN, S., BUTCHER, G. A. and CRANDALL, R. B. (1969). 'Action of malarial antibody in vitro.'

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Vaccination against Malaria GARNHAM, P. C. C. (1966). Malaria Parasites, p. 332. Blackwell Scientific Publications, Oxford. MITCHELL, G. H., BUTCHER, G. A. and COHEN, S. (1973). 'Isolation of blood-stage merozoites from Plasmodium knowlesi malaria.' Int. J. Parasit., 3, 443. MITCHELL, G. H., BUTCHER, G. A. and COHEN, S. (1974). 'A merozoite vaccine effective against Plasmodium knowlesi malaria.' Nature (Lond.), 252, 311. SADUN, E. H., HICKMAN, R. L., WELLDE, B. T., MOON, A. P. and UDEOZO, I. 0. K. (1966). 'Active and passive immunisation of chimpanzees infected with West African and South-east Asian strains of Plasmodium falciparum.' Milit. Med. 131, 1250. SCHENKEL, R. H., SIMPSON, G. L. and SILVERMAN,


P. H. (1973) 'Vaccination of rhesus monkeys (Macaca mulatta) against Plasmodium knowlesi by the use of nonviable antigen.' Bull. WId. HIth Org., 48, 597. SIMPSON, G. L., SCHENKEL, R. H. and SILVERMAN, P. H. (1974). 'Vaccination of rhesus monkeys against malaria by use of sucrose density gradient fractions of Plasmodium knowlesi antigens.' Nature (Lond.), 247, 304. TARGETT, G. A. T. and FULTON, J. D. (1965). 'Immunisation of rhesus monkeys against Plasmodium knowlesi malaria.' Exp. Parasit. 17, 180. VOLLER, A. and ROSSAN, R. N. (1969). 'Immunological studies on simian malaria. III. Immunity to challenge and antigenic variation in P. knowlesi.' Trans. roy. Soc. trop. Med. Hyg., 63, 507.

Merozoite vaccination against Plasmodium knowlesi malaria.

Free malarial merozoites isolated from in vitro cultures of P. knowlesi and emulsified with Freund's complete (FCA) or incomplete (FIA) adjuvant were ...
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