A review of merozoite vaccination against Plasmodium knowlesi malaria G. H. MITCHELL Department of Chemical Pathology, Guy’s Hospital Medical School, London SE1 9RT

Summary Techniques for the isolation of merozoites of Plasmedium knowlesi malaria have allowed their use in experimental vaccines. Rhesus monkeys were protected to a very great extent from otherwise lethal challenge with this malaria when Freund’s Complete Adjuvant was a vaccine component. Introduction Studies in vivo in children naturally infected with Plasmodium falcigavum (COHEN& MCGREGOR,1963) and in vitro on P. knowlesi from rhesus monkeys (COHEN et al., 1969) suggested that antibody capable of inhibiting merozoite invasion of red cells was an important component in the protective immune response to malaria. This prompted efforts to isolate erythrocytic merozoites of P. knowlesi for use in the experimental vaccination of rhesus monkeys. Isolation of Merozoites P. knowlesi parasites in the rhesus monkey show synchronous development and very high termimal parasitaemias are characteristic. Thus donor monkeys infected by injection of lo4 asexual parasites were bled after seven or eight days when a large proportion (normally 20 to 70% of red cells were oarasitized. and schizonts with six 0; eight nuclei predominated. After gentle centrifugation (350 x g, 8 minutes) schizontinfected cells formed a brown layer lying between the huffy coat and uninfected erythrocytes, and wereaspirated off largely uncontaminated with normal red cells. Two methods were developed for the collection of merozoites. Incubation of dilute suspensions of schizonts (about lO’/ml) in modified medium 199 (MITCHELL et al., 1973) at 37”C, in suitable flasks under a 5% COZ atmosphere, resulted in segmentation and rupture of the parasites. Free merozoites accumulated since almost no normal red cells were available for invasion. The addition of phytohaemagglutinin (PHA Reagent Grade, Wellcome; about 1.5 mg/ml culture suspension) (MITCHELL et al., 1975) or, in earlier experiments, schizont agglutinating antiserum variant-specific (MITCHELL et al,, 1973), caused massive agglutination of unruptured schizont-infected cells, facilitating their precipitation by gentle centrifugation (250 x g, 10 minutes) leaving a supernatant rich in merozoites contaminated by few (< 0.1%) host cells. Alternatively, schizont-infected cells were incubated in the Cell Sieve, a specially designed culture chamber incorporating a polycarbonate membrane of 2~ pore-size through which medium could be pumped continuously (DENMS et al., 1975). As merozoites were released from rupturing schizonts they were swept from the chamber

in the medium flow through the membrane whilst unruptured schizonts were retained. Very high yields of merozoites, approaching the theoretical mean maximum of x 10 the number of schizonts introduced into the apparatus, were sometimes achieved during several hours operation (E. D. DENNIS, unpublished observation). Vaccination of Monkeys Merozoites of either provenance have been used as antigen without apparent differences in efficacy. The viability of extra-cellular merozoites is of very limited duration (DENNIS et al., 1975), and no specific action was taken to render merozoites non-infective before use as antigen. Monkeys were immunized with either freshly prepared merozoites, or with merozoites stored deep frozen at - 70” in saline, or stored freeze-dried at 4” for periods of up to three months (MITCHELL et al., 1977). Exposure to 1:lOOO formalin in saline overnight at 4” before freeze-drying severely reduced the immunogenicity of merozoites (BUTCHER et al., 1977) (Table I). Two, three or, on two occasions, four doses of merozoites were given, each containing between 5 x 10’ and 5 x lo9 merozoites (mean dose 1.9 x 109)over periods of 22 to 98 days. Merozoites were derived from a single antigenic Table I - Challenges of merozoites Nature of Challenge

Blood: Homologous variant Heterologous variant Heterologous strain Sporozoites: Heterologous strain

monkeys immunized


Results of Challenge : Survivors/Total Immunized using Otherwise FCA on 2 or immunized more occasions SubSubInitial sequent Initial sequent 10/13*















* Deaths in monkeys given formolized merozoites. ** Three deaths in monkeys given formolized merozoites. t l/l FCA:FTA, l/3 (BCG) FIA:FIA, O/l Alhydrogel, O/l Adjuvant 65. tt O/l FCA:FIA, O/2 (BCG) FIA:FIA, O/l Alhydrogel.





Table II-Initial Vaccine

Challenges of Monkeys Vaccinated twice or more with Merozoites* in FCA Initial Challenge Survivors No. Strain Stage Parasitaemia No. Monkeys (variant) Monkeys Negative Maximum %

Fresh Fresh Fresh Freeze-dried Freeze-dried Frozen

5 6 2 2 4 2

w w A w w w

(1) (3) (1) (3) (3)

Blood Blood Sporozoite Blood Blood Blood

5 5 2 2 4 2

2 0 0 0 2 1

0.04-l .s 0.01-0.3 0.04-0.7 0.40-5.9 0.60-3.7 0.6

Died Duration (days) 6-13 1-21 9-11 16-17 11-18 11

0 1 0 0 0 0

* Excludes results from monkeys which received formaldehyde-treated merozoites. variant (BROWN & BROWN, 1965) designated WI (BUTCHER& COHEN,1972). Vaccine was prepared by the admixture of merozoites and adjuvant and given by intramuscular injection. Five adjuvants have been investigated alone or in combination: Freund’s Complete Adjuvant (FCA) and Freund’s Incomplete Adjuvant (FIA) (Difco); Adjuvant 65-4 (Merck, Sharp & Dohme); Alhydrogel (Wellcome); BCG (Glaxo). Of these BCG (lo6 organisms) was given as a priming dose to animals which later received merozoites in FIA, and Alhydrogel was employed only with merozoites pretreated with formaldehyde. Parasites used for Challenge Infections Blood-induced challenge infections were of five variants of the parasite strain used for vaccination serologically defined by the SICA test (BROWN& BROWN, 1965), and one variant of another strain (W and N strains; see MITCHELL et al., 1975). Initial blood challenges were usually with lo4 late trophozoites or early schizonts but for subsequent challenges much larger numbers of parasites, up to lo”‘, have been injected (BUTCHERet al., 1977). Sporozoite challenges were of the A strain of P. knowlesi and were administered either by the feeding of infected Anopheles balabacensis or by the intravenous injection of 2 x lo3 parasites dissected from mosquito salivary glands (RICHARDSet al., 1977). Blood-induced infections of P. knowlesi are nearly uniformly fatal in normal rhesus monkeys, death occurring six to 11 days after infection with lo4 parasites, whereas sporozoite-induced infections of A strain P. knowlesi may follow a chronic and relatively benign course (L. H. MILLER, personal communication). Sporozite-induced infections killed three of six control animals in these studies; the surviving animals remained patent until drug-cured five or six weeks after infection (RICHARDSet al., 1977). Results of Challenges Results of initial challenges of monkeys immunized twice or more with merozoites not previously formoltreated in FCA are summarized in Table II. All survived homologous variant challenge, two without patent infection. Parasitaemias were of brief duration and generally low intensity. Initial heterologous variant challenge of twelve animals killed one whilst two remained parasitologically negative; patent survivors cleared parasitaemias within three weeks. On several occasions blood from a vaccinated monkey was subinoculated into a normal monkey after a challenge infection had been resolved. The absence of infection in the recipients suggested that vaccinated monkeys exhibited a sterilizing response. Sporozoite-induced

A-strain challenges were similarly cleared. A total of 42 subsequent challenges of all specificities were administered to the surviving monkeys, only one resulting in a lethal infection (Table I). Immunity to challenge remained solid for at least two years after vaccination in a repeatedly challenged animal, and for at least five months between challenges (RICHARDSet aZ., 1977). None of the other adjuvants tested in the merozoite vaccine has produced such nearly consistent immunity as has Freund’s Complete Adjuvant given on two occasions (Table I). The unacceptability of this adjuvant for clinical use and the difficulty of replacing it remain chief constraints on the development of a vaccine against human malaria. References Brown, K. N. & Brown, I. N. (1965). Immunity to malaria: antigenic variation in chronic infections of Plasmodium knowlesi. Nature, 208, 1286-1288. Butcher, G. A. & Cohen, S. (1972). Antigenic variation and protective immunity in Plasmodium knowlesi malaria. Immunology, 23, 503-521. Butcher, G. A., Mitche!l, G. H. & Cohen, S. (1977). Merozoite vaccination against Plasmodium knowlesi malaria: mechanisms of induced immunity. (In preparation.) Cohen, S., Butcher, G. A. & Crandall, R. B. (1969). Action of malarial antibody in vitro. Nature, 223, 368-371. Cohen, S. & McGregor, I. A. (1963). y-Globulin and acquired immunity to malaria. Inf Zmmunity to Protozoa, Garnham, P. C. C., Pierce, A. E. & Roitt, I. (Editors). Oxford: Blackwell Scientific Publications, pp. 123-159. Dennis, E. D., Mitchell, G. H., Butcher, G. A. & Cohen, S. (1975). In vitro isolation of Plasmodium knowlesi merozoites using polycarbonate sieves. Parasitology, 71,475-481. Mitchell, G. H., Butcher, G. A. & Cohen, S. (1973). Isolation of blood-stage merozoites from Plasmodium knowlesi malaria. International Journal for Parasitology, 3, 443445. Mitchell, G. H., Butcher, G. A. & Cohen, S. (1975). Merozoite vaccination against Plasmodium knowlesi malaria. Immunology, 29, 397407. Mitchell, G. H., Butcher, G. A., Langhorne, J. & Cohen, S. (1977). A- freeze-dried merozoite vaccine effective aaainst Plasmodium knowlesi malaria. Clinical and E;perimental Immunology. (In press.) Richards, W. H. G., Mitchell, G. H., Butcher, G. A. & Cohen, S. (1977). Merozoite vaccination of rhesus knowlesi malaria: monkeys against Plasmodium immunity to sporozoite (mosquito-transmitted) challenge. Parasitology, 74,191-198. .


A review of metozoite vaccination against Plasmodium knowlesi malaria.

281 TRANSACTIONSOF THE ROYAL SOCIETYOF TROPICAL MEDICINE AND HYGIENE, VOL. 71, No. 4,1977 A review of merozoite vaccination against Plasmodium knowle...
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