Clin. exp. Immunol. (1977) 28, 276-279.

A freeze-dried merozoite vaccine effective against Plasmodium knowlesi malaria G. H. M ITCHELL, G. A. BUTCHER, J. LANGHORNE & S. COHEN Department of Chemical Pathology, Guy's Hospital Medical School, London (Received 8 December 1976)

INTRODUCTION Malaria eradication measures have proved ineffective in many tropical areas. The disease currently has an estimated world-wide morbidity of 100 million cases and mortality of more than 1 million per annum, mainly among children (editorial Lancet, 1975). Under these circumstances alternative methods of control, including vaccination, are of obvious interest. Plasmodium knowlesi malaria, when experimentally transmitted by blood infection, is rapidly fatal in rhesus monkeys. If infection is combined with subcurative drug therapy, animals become partially immune and develop chronic relapsing infections, each relapse being associated with serologically distinct parasite populations recognized by a schizont-infected cell agglutination (SICA) test (Brown & Brown, 1965). Antigenic variation appears to be one factor determining chronicity of infection and must be taken into account in evaluating attempted immunoprophylaxis (Cohen, 1974). Vaccination of rhesus monkeys with inactivated parasitized red cells or extracts from these in Freund's complete adjuvant (FCA) reduces mortality to about 50% after challenge with the immunizing variant of P. knowlesi but provides no protection against challenge with a different variant (Brown, 1976; WHO Technical Report, 1975). Studies on the mechanism of acquired malarial immunity in vivo and in vitro indicated that protective antibody interrupts parasite development at the stage when merozoites are released into the plasma (Cohen, Butcher & Mitchell, 1974). Immune sera prevent merozoite attachment to receptors present on the red cells of susceptible species (Butcher, Mitchell & Cohen, 1973; Miller et al., 1973). These findings indicated an important role for merozoite antigens in specific malarial immunity and prompted development of methods for isolating extra-cellular blood stage merozoites (Mitchell, Butcher & Cohen, 1973; Dennis, Mitchell, Butcher & Cohen, 1975). Rhesus monkeys vaccinated with merozoites in Freund's complete adjuvant are protected against blood or mosquito-borne (sporozoite) challenge with either the immunizing variant or other variants or strains of P. knowlesi (Mitchell et al., 1975; Richards et al., 1977). In the present study, the effectiveness of a freeze-dried merozoite vaccine in protecting against homologous or heterologous variant blood challenge is demonstrated. MATERIALS AND METHODS Imported rhesus monkeys (Macaca mulatta) of either sex weighing 2-4 kg were caged in a room artificially illuminated between 7.00 a.m. and 7.00 p.m. The P. knowlesi parasite used was a Malaysian strain designated W (Butcher & Cohen, 1972) originally obtained from the Walter-Reed Army Institute of Research, Washington. Serologically distinct variants of this strain were identified by the schizont-infected cell agglutination (SICA) test (Brown & Brown, 1965). Variants maintained their specificity unchanged after storage at - 80'C in 15% glycerol-saline or during repeated passage through normal rhesus monkeys (Butcher & Cohen, 1972). Parasite donors or unimmunized controls were cured as necessary with chloroquine and sulphadiazine as described (Butcher & Cohen, 1972). Monkeys were vaccinated with WI-strain merozoites isolated from in vitro cultures by differential precipitation with phytohaemagglutinin (Mitchell et al., 1975). Within 3 hr of isolation these organisms were suspended in phosphate-buffered

Correspondence: Dr G. H. Mitchell, Department of Chemical Pathology, Guy's Hospital Medical School, London SEl.

276

Freeze-dried merozoite vaccine

277

TABLE 1. Results of homologous (W1) and heterologous (W3) challenge of three normal monkeys and eight vaccinated with freeze-dried or frozen W1-merozoites in FCA

Merozoite vaccine Monkey no. Mz 61 Mz 62 Mz 57 Mz 58 Mz 63 Mz 64 Mz 59 Mz 60 519 510 520

Treatment

f.d. f.d. f.d. f.d. f.d. f.d. Fz. Fz.

Total no. x 109

4 4 3-5 3-5 4 4 35 3-5

Challenge: Days*

Dayt

Parasite

Pre-patent (days)

Duration (days)

Max/104 RBC

1,22 1,22 1,25 1,25 1,22 1,22 1,25 1,25

49 49 54 54 56 56 54 54 1 1 1

WI Wi W3 W3 W3 W3 W3 W3

6 7

16 17

50 590

W1

W3 W3

Negative Negative 4 4

18 13

6 2 3 4

12

260 370

Negative

45 4§

45

60 2500 5500 1200

f.d. = Freeze-dried (see the Materials and Methods section); Fz. = frozen. * Days on which vaccine was injected. t Day of challenge (after first vaccination) or day of infection of normal controls. + Challenge = I.VI. 104 schizonts for vaccinated. Controls received 104 parasites (519) or glycerol-stored parasites. § Died.

saline, snap-frozen in liquid N2 and stored at - 80°C; alternatively, merozoites were freeze-dried from suspension in 10% normal rhesus serum in water. The frozen and freeze-dried vaccines were stored for up to 12 weeks before use. Vaccine was emulsified in FCA before injection (Mitchell et al., 1975) and monkeys inoculated i.m. on two occasions at intervals of 3-4 weeks; a total of about 4 x 109 merozoites was administered to each monkey (Table 1). Vaccinated monkeys were challenged by i.v. injection of 104 immature schizonts of known variant specificity taken from infected donors which also served as normal controls (Table 1).

RESULTS All monkeys were vaccinated with merozoites derived from the W1 variant of P. knowlesi. Two variants, W1 and W3, were used for blood induced challenge. Normal monkeys developed rapidly increasing parasitaemia and died on the 6th to 8th day after inoculation of either variant (Table 1). Two monkeys vaccinated with freeze-dried merozoites and challenged with the homologous variant (W1) developed infections (maximum parasitaemia 05o% and 5 9%) which terminated after 16 or 17 days (Table 1). A further four rhesus vaccinated with freeze-dried merozoites were challenged with a heterologous variant (W3). Two did not develop patent infections and two showed parasitaemia (maximum levels 2-6 and 3 7%/) which lasted 13 and 18 days, respectively (Fig. 1). The remaining two animals were vaccinated with merozoites stored in the frozen state and were challenged with the W3 variant; one showed no parasitaemia and the other developed an infection which lasted 12 days (maximum 0.6%) (Table 1 and Fig. 1). These results indicate that merozoites stored by freeze drying or in the frozen state are as effective as freshly isolated merozoites when used for vaccination in conjunction with FCA. With both freshly isolated and stored vaccines all animals survived homologous (W1) challenge. After vaccination with freshly isolated merozoites two out of twelve animals succumbed to first challenge with a heterologous variant or strain of P. knowlesi (Table 2); parasitaemia did not exceed 2-9%/ in the surviving animals and infections terminated after 1 to 21 days. Here six monkeys vaccinated with frozen or freeze-dried merozoites all survived heterologous challenge and three did not become patent; infections in the remaining three reached 00i3-7o% and lasted for 12-18 days. F

278

G. H. Mitchell et al.

21-0 170

I Died -

13-0 .X

90

._ E

m

5-0-

0m

L

10 0-5 _

20 25 10 15 Days after challenge FIG. 1. Course of parasitaemia in one normal rhesus and three monkeys vaccinated with freeze-dried (Mz 63 and 64) or frozen (Mz 60) W,-variant merozoites in FCA. The monkeys were challenged with 104 W3-variant schizonts of P. knowlesi by i.v. inoculation on day 0. Two other monkeys vaccinated with freeze-dried merozoites did not develop patent infections (Table 1). The vertical scale is interrupted to illustrate the course of low grade parasitaemia in vaccinated animals. (o) Mz 60; (C) Mz 63; (0) Mz 64; (O) control.

5

0

TABLE 2. Vaccination with P. knowlesi merozoites (W1) in FCA. Summary of results of first challenge by blood or mosquito-induced (sporozoite) infection

Died No. of monkeys

F* F* Ft F.D. F.D. Fz

Survived challenge

Challenge

Merozoite vaccine

5 6 6 2 4 2

Strain (variant) W (1) W (3) A W (1) W (3)

W(3)

Stage

Sz Sz

Sp Sz Sz Sz

No. of monkeys

5 5 5 2 4 2

Negative

2 0 0 0 2 1

Parasitaemia Maximum Duration

(%)

(days)

0 041-8 0-01-0-3 0-01-2-9 0-50-5-9

6-13 1-21 1-20 16-17 12-18 12

0-60-37 06

0 1 1 0 0 0

F = Freshly prepared; Fz = frozen; F.D. = freeze-dried (see the Materials and Method; section); Sz = immature schizonts; Sp = sporozoites. * Mitchell et al. (1975). t Richards et al. (1977).

Freeze-dried merozoite vaccine

279

DISCUSSION P. knowlesi is almost invariably fatal in the rhesus monkey so that this host parasite combination provides the most stringent of all models available for testing a malaria vaccine. Experience ofmerozoite vaccination has been confined to this malarial infection in the rhesus (Mitchell et al., 1975; WHO Technical Report, 1975). The vaccine does not require inactivation since merozoites are no longer viable after storage for 1 hr (Dennis et al., 1975). The induced immunity lasts for at least 6 months and is directed against the blood stage parasite which is responsible for clinical manifestations of malaria. Development of the pre-erythrocytic stage which follows mosquito inoculation of sporozoites is not inhibited by merozoite vaccination but the subsequent erythrocytic infection is terminated (Richards et al., 1977). Immunity is species specific, but has broad variant and strain specificity (Mitchell et al., 1975; Richards et al., 1977). In contrast to the chronic relapsing parasitaemia which occurs with the natural evolution of immunity or after repeated infection and drug cure, merozoite vaccinated animals eliminate all viable blood parasites after a brief infection of low intensity (Mitchell et al., 1975). The possibility of producing merozoites for mass vaccination has become plausible following the recent successful cultivation ofP. fakiparum in human erythrocytes (Trager & Jensen, 1976; Haynes, Diggs, Hines & Desjardins, 1976). The practicability of vaccination is enhanced by the present demonstration that freeze-dried merozoites constitute an effective vaccine. The requirement for Freund's complete adjuvant remains a constraint on the development of a merozoite vaccine suitable for human use. Other adjuvants have proved less effective (Butcher, Mitchell & Cohen, in preparation) but further alternatives for use with merozoite vaccination are under investigation. This work is supported by the Medical Research Council, London, and the World Health Organization. We are indebted to Miss Marilyn Burnikell for skilled technical help. REFERENCES BROWN, K.N. (1976) Resistance to malaria, Immunology of EDITORIAL (1975) Epitaph for global malaria eradication ? Parasitic Infections, (ed. by S. Cohen and E.H. Sadun), p. Lancet, ii, 15. 261. Blackwell Scientific Publications, Oxford. HAYNES, J.D., DIGGS, C.L., HINEs, F.A. & DESJARDINS, R.E. BROWN, K.N. & BROWN, I.N. (1965) Immunity to malaria: (1976) Culture of human malaria parasites Plasmodium antigenic variation in chronic infections of Plasmodium falciparum. Nature (Lond.), 263, 767. knowlesi. Nature (Lond.), 208, 1286. MILLER, L.H., DvoRAK, J.A., SHIROISHI, T. & DUROCHER, BUTCHER, G.A. & COHEN, S. (1972) Antigenic variation and J.R. (1973) Influence of erythrocyte membrane compoprotective immunity in Plasmodium knowlesi malaria. nents on malaria invasion. J. exp. Med. 138, 1597. Immunology, 23, 503. MITCHELL, G.H., BUTCHER, G.A. & COHEN, S. (1973) BUTCHER, G.A., MITCHELL, G.H. & COHEN, S. (1973) Isolation of blood stage merozoites from Plasmodium Mechanisms of host specificity in malarial infection. knowlesi malaria. Int. J. Parasitol. 3, 443. Nature (Lond.), 244, 40. MITCHELL, G.H., BUTCHER, G.A. & COHEN, S. (1975) COHEN, S. (1974) Immunophophylaxis of protozoal diseases, Merozoite vaccination against Plasmodium knowlesi Clinical Aspects of Immunology, 3rd edn (ed. by R.G.H. malaria. Immunology, 29, 397. Gell, R.R.A. Coombs and P.J. Lachmann), p. 1649. RICHARDS, W.H.G., MITCHELL, G.H., BUTCHER, G.A. & Blackwell Scientific Publications, Oxford. COHEN, S. (1977) Merozoite vaccination of rhesus COHEN, S., BUTCHER, G.A. & MITCHELL, G.H. (1974) monkeys against Plasmodium knowlesi malaria; immunity Mechanisms of immunity to malaria. Bull. Wld HIth Org. to sporozoite (mosquito-transmitted) challenge. Para50, 251. sitology, (In press.) DENNIS, E.D., MITCHELL, G.H., BUTCHER, G.A. & COHEN, TRAGER, W. & JENSEN, J.B. (1976) Human malaria parasites S. (1975) In vitro isolation of Plasmodium knowkesi in continuous culture. Science, 193, 673. merozoites using polycarbonate sieves. Parasitology, 71, WORLD HEALTH ORGANISATION (1975) Developments in 475. malaria immunology. Technical Report series no. 579.