Biomedical subjects
Publications and source records attributed to G A Butcher.
A human malaria vaccine that will protect against the blood stages of Plasmodium falciparum is dependent upon the continuous cultre of the parasite. Scientists have demonstrated that it is now possible to achieve this on a small scale. Present expertise needs to be expanded to produce larger quantities of material for antigenic studies and potential vaccine production.
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Previous observations on the process of invasion by P. knowlesi are briefly reviewed and new findings concerning the adhesion of parasites to erythrocytes and their intracellular passage are reported. Merozoites adhere to erythrocytes by means of a well-defined coat with the cytochemical characteristics of glycoprotein. This coat has receptors that engage in at least three distinct types of attachment. The ensuing invagination of the erythrocyte surface has two phases, the first consisting of an inward buckling of the membrane to form a vacuole and the second a rapid expansion to create the final parasitophorous vacuole inhabited by the trophic parasite. Some cytochemical evidence concerning the nature of the rhoptry complex is discussed in relation to these changes.
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Freeze-fracture studies were made on the parasite and the erythrocyte in P. knowlesi infections. There is a loss of transmembrane integral proteins from the plasma membrane of the schizont-infected erythrocyte and the intraerythrocytic parasite synthesizes new transmembrane proteins as development proceeds. Formation of the parasitophorous vacuole includes changes in the number of integral proteins present in the vacuolar membrane, indicating that this membrane may be modified by and in part derived from the parasite.
Plasmodium falciparum parasites infecting Aotus trivirgatus erythrocytes were cultured in media (Harvard and TC199) augmented with human, foetal calf, or other sera. Conditions were established which supported growth of parasites and allowed cyclical multiplication when fresh erythrocytes (from Aotus or Homo) were added in sub-culture (mean multiplication rate: X3). Immunoglobulin G pools, prepared from plasma collected in endemic malarious areas in Africa and from unexposed Britons, were tested for effects on the in vitro growth (measured by incorporation of tritiated leucine) and multiplication of parasites. Whilst non-immune IgG was without effect, IgG from both East and West Africa inhibited the multiplication of East African (Uganda-Palo Alto strain) parasites.
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A culture chamber fitted with a polycarbonate sieve has been used to isolate Plasmodium knowlesi merozoites as they are released from schizonts. A 3 mum pore-size sieve allows passage of normal erythrocytes and red cells containing rings and trophozoites and can be used to concentrate schizonts from a mixed cell population. A 2 mum pore-size sieve retains normal and parasitized cells and provides uncontaminated merozoites in high yield (5 x 10(10) merozoites per ml schizonts). Merozoite viability diminishes rapidly during 30 min after isolation. These preparations should prove valuable for studies of the biochemical, physiological and antigenic properties of this transient phase of the malaria parasite.
The structure and invasive behaviour of extracellular erythrocytic merozoites prepared by a cell sieving method have been studied with the electron microscope. Free merozoites contain organelles similar to those described in late schizonts of Plasmodium knowlesi. Their surface is lined by a coat of short filaments. On mixing with fresh red cells, merozoites at first adhere, then cause the red cell surface to invaginate rapidly, often with the formation of narrow membranous channels in the red cell interior. As the merozoite enters the invagination it forms an attachment by its cell coat to the rim of the pit, and finally leaves this coat behind as it is enclosed in a red cell vacuole. Dense, rounded intracellular bodies then move to the merozoite periphery, and apparently rupture to cause further localized invagination of the red cell vacuole. The merozoite finally loses its rhoptries, the pellicle is reduced to a single membrane and the parasite becomes a trophozoite. Invasion is complete by 1 min after adhesion, and the trophozoite is formed by 10 min.
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 1.5 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 1.5 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.
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The erythrocytic phase of malarial infection provides a potent stimulus for the production of specific malarial antibody. Serologic tests do not provide any indication of immune status, so that much specific antibody has no protective function. The role of serum antibody in acquired malarial immunity has, however, been established by passive transfer tests, and in the case of P. knowlesi by specific inhibition of the cyclic growth of parasites in vitro. The inhibitory antibody appears to combine with merozoites and prevents their attachment to red cells, thus interrupting the cyclic proliferation of the parasite. The inhibitory antibody response is predominantly variant-specific, but cross-reacting antibody occurs in sufficient amount to suppress proliferation of most other variants of the species. The occurrence of cross-immunity between variants is encouraging from the point of view of vaccination. If it were possible to isolate cross-reacting antigens, these could provide the basis for a malarial vaccine effective against erythrocytic forms of the parasite.
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Explore the source record for details and available documents.
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