Cultivation techniques for the erythrocytic stages of malaria parasites

The study of the biochemistry, physiology, and immunology of plasmodia has been restricted by the difficulty of maintaining the parasites in isolation from the host. Some success has been achieved in cultivating them in vitro, using tissue cultures and chick embryo techniques to study exoerythrocytic states and the sporogonic cycle, but no completely successful method has been found for studying the asexual and sexual stages of plasmodia in circulating red blood cells. The relative slowness with which techniques for continuous in vitro cultivation have been developed is the result of inadequate knowledge of the biochemistry of the parasites and of the blood and its constituents. However, radioactive labelling techniques applied to P. knowlesi cultures are beginning to yield data of fundamental importance. Existing methods for the short-term in vitro cultivation of plasmodia are potentially very useful for analysing malarial antigens, for developing vaccines, and for screening and studying antimalarial drugs. Investigations of the physicochemical requirements for the in vitro preservation of red blood cells are required, and more emphasis should be given to the study of plasmodia with longer cycles. Differences between the metabolism of plasmodia in vivo and in vitro should be studied and the growth factors in normal plasma identified. Studies of the membrane of the parasites and of the red blood cells, of the immune response, and of extracellular methods for the cultivation of plasmodia should be extended.     

Recent advances in malaria parasite cultivation and their application to studies on host-parasite relationships: a review

The continuous cultivation of the erythrocytic stages of Plasmodium falciparum was achieved in 1976 and techniques have now also been developed for continuous cultivation of these stages from P. knowlesi, P. fragile, P. inui, P. cynomolgi and P. berghei. The requisite conditions for successful cultivation are described. Gametes of certain isolates of P. falciparum can also now be produced in vitro and these are infective to mosquitos, leading to normal development of the parasite.     

The interaction between sickle haemoglobin and the malarial parasite Plasmodium falciparum

The mechanism whereby heterozygous carriers of the sickle cell gene are protected against fatal malarial infections due to Plasmodium falciparum has been examined in a short term in vitro cultivation system. The results have show that both parasite invasion of red cells and parasite growth within red cells containing sickle haemoglobin (Hb-S) is restricted, but only under conditions of low (5%) oxygen tensions. To bring this about, the cells containing Hb-S need not sickle. Furthermore the growth retardation observed in the presence of Hb-S was also found to apply to the mature forms of the parasite. These findings offer a plausible mechanism for the protection of sickle heterozygotes against falciparum malaria.     

Recent progress in the development of malaria vaccines: Memorandum from a WHO Meeting

The sixth meeting of the Scientific Working Group on the Immunology of Malaria reviewed studies on the identification and analysis of malarial antigens of asexual blood stages and sexual stages (gametes, zygotes, ookinetes) that may be exploited as targets for vaccination. Several proteins have been identified on the surface of mature schizonts and free merozoites, some of which can be recognized by antibodies which block in vitro parasite growth. Immunization of rodents and monkeys with purified antigens from the parasite surface membrane has conferred substantial immunity against subsequent challenge. A new class of malarial antigens has been identified which bind specifically to glycophorin, the major erythrocyte glycoprotein; these antigens are on the merozoite surface and it is possible that they mediate attachment to erythrocytes. Antibodies against these proteins also block parasite growth in vitro. The Plasmodium falciparum S-antigens have been characterized biochemically and the genes for two of these proteins sequenced. Several antigens have been localized in the invasion process, and monoclonal antibodies against these proteins block in vitro growth. Malarial antigens on the surface of P. falciparum trophozoite and schizont-infected erythrocytes may be involved in the cytoadherence of infected erythrocytes to endothelial cells. Surface antigens on gametes and zygotes of P. gallinaceum and P. falciparum have been shown to be the targets of transmission-blocking immunity. Monoclonal antibodies specific for these antigens block fertilization in the mosquito midgut. Transmission of P. gallinaceum can also be blocked by an antibody that blocks development of zygotes into ookinetes. Studies on the transmission of P. yoelii have identified a gamete protein that immunizes mice against transmission to mosquitos.     

Development of malaria vaccines: Memorandum from a USAID/WHO meeting

The fifth meeting of the Scientific Working Group on the Immunology of Malaria evaluated studies of the production and analysis of defined malarial antigens. Rapid progress has been made in the study of protective antigens on the surface of sporozoites and it is likely that a family of analogous polypeptides occurs in several species of Plasmodium. New assays have been developed for the detection of these antigens and for the detection of infected mosquitos. Exoerythrocytic stages of several parasite species can be cultivated in vitro, providing an assay system for antibody and allowing the characterization of exoerythrocytic stage antigens. Progress has also been made in the identification of species- and stage-specific antigens of the asexual blood stages of rodent, simian, and human malaria parasites. In some instances, protective immunity has been shown to be directed against polypeptides (with a high relative molecular mass) synthesized at a late stage of schizont development. Messenger RNA (mRNA) species from P. knowlesi and P. yoelii have been successfully translated in vitro to give polypeptides with a high relative molecular mass (M_r). Monoclonal antibodies have been used to identify and to purify important parasite antigens and purified P. yoelii antigens induced protective immunity. Monoclonal antibodies reactive with merozoite surface antigens have been used, as well as S-antigens, to distinguish between different isolates of P. falciparum. Recombinant DNA technology is being applied to Plasmodium: differences were found between repetitive DNA sequences from the genome of two isolates of P. falciparum; the genes for ribosomal RNA of P. falciparum and P. yoelii, and sequences homologous to the actin gene were identified in fragments of Plasmodium DNA cloned in prokaryotic vectors; by means of hybrid selection, complementary DNA (cDNA) probes were used to purify mRNAs encoding proteins of P. knowlesi of up to 100 000 M_r.     

Malaria parasite strain characterization, cryopreservation, and banking of isolates: a WHO Memorandum

There has been considerable progress in the biological characterization of malaria parasites in the past few years. Physiological parameters such as host adaptation, virulence, exoerythrocytic development, in vitro growth of erythrocytic stages, and drug sensitivity are of particular importance to epidemiologists. Advances in enzyme analysis, 2-dimensional protein electrophoresis, and nucleic acid analysis have produced several new techniques that can be applied to the malaria parasite. Similarly, antigenic characterization is expected to progress as a result of technical improvements. Many of the biological parameters are needed for the study of parasite genetics, a field which has expanded greatly through the development of cloning techniques. The latter also hold interest for the production, and the future use in research, of biologically well characterized standard clones. In this connexion, the cryopreservation and banking of malaria parasites deserve attention, in order to ensure the supply of well defined, viable isolates and clones to interested research workers.     

Diagnosis of Plasmodium falciparum infection using a solid-phase radioimmunoassay for the detection of malaria antigens

A serodiagnostic test has been developed for the detection of Plasmodium falciparum in infected blood. Using parasite antigens and infected red blood cells from in vitro cultures of P. falciparum and malaria antibody from high-titre Gambian sera, parasites were detected in a solid-phase radioimmunoassay (RIA) that measured antibody-binding inhibition. Lysed RBC were incubated with labelled IgG purified from immune sera and were then placed in antigen-coated microtubes and incubated. The degree of inhibition of antibody binding in the tubes correlated with the level of parasitaemia in the test RBC and, using dilutions of RBC from in vitro cultures of P. falciparum, the test detected infection at a level of 8 parasites/10⁶ red blood cells. The test was applied to RBC from 100 healthy European blood donors and to samples of RBC from 500 Gambians from the up-country villages of Keneba and Manduar. More samples were positive by RIA than by microscopy and there was a highly significant degree of correlation between the RIA and microscopy results.     

Growth of protozoa in tissue culture. III. Trypanosoma cruzi

The technique used for cultivation of exoerythrocytic forms of P. gallinaceum has been applied to the growth of Trypanosoma cruzi also. A suspension of trypanosomes from the blood of an infected mouse was added to cultures of rat embryo. During the first 2 days great numbers of trypanosomes were present in the culture fluid, not particularly associated with the cells. Then they gradually became fewer until by about the 12th day they were rare or absent. Later small numbers reappeared in the culture fluid and persisted there until the end of the culture. Stained preparations made after. 6 days or more showed great numbers of intracellular parasites in all stages between the rounded leishmanoid form and the mature trypanosome form. The parasite occurred in cardiac muscle fibres, in macrophages and in elongated cells with processes (probably reticulo-endothelial cells). Cultures have been maintained for 59 days. As described by Meyer (1942) T. cruzi could also be grown in cultures from the brain of chick embryo.This technique offers favourable conditions for study of the intracellular development of T. cruzi and of the action of drugs upon it.     

Effects of study area size on geographic characterizations of health events: Prostate cancer incidence in Southern New England, USA, 1994–1998

Background

In this paper we analyse the Plasmodium sp. prevalence in three villages with different isolation status on the island of Bioko (Equatorial Guinea) where malaria is a hyper-endemic disease. We also describe the genetic diversity of P. falciparum, using several plasmodia proteins as markers which show a high degree of polymorphism (MSP-1 and MSP-2). The results obtained from three different populations are compared in order to establish the impact of human movements and interventions.

Methods

Plasmodium sp. were analysed in three villages on Bioko Island (Equatorial Guinea), one of which (Southern) is isolated by geographical barriers. The semi-nested multiplex polymerase chain reaction (PCR) technique was used to determine the prevalence of the four human plasmodia species. The genotyping and frequency of P. falciparum populations were determined by PCR assay target polymorphism regions of the merozoite surface proteins 1 and 2 genes (MSP-1 and MSP-2).

Results

The data obtained show that there are no differences in plasmodia population flow between the Northwest and Eastern regions as regards the prevalence of the different Plasmodium species. The Southern population, on the other hand, shows a minor presence of P. malariae and a higher prevalence of P. ovale, suggesting some kind of transmission isolated from the other two. The P. falciparum genotyping in the different regions points to a considerable allelic diversity in the parasite population on Bioko Island, although this is somewhat higher in the Southern region than the others. There was a correlation between parasitaemia levels and the age of the individual with the multiplicity of infection (MOI).

Conclusion

Results could be explained by the selection of particular MSP alleles. This would tend to limit diversity in the parasite population and leading up to the extinction of rare alleles. On the other hand, the parasite population in the isolated village has less outside influence and the diversity of P. falciparum is maintained higher. The knowledge of parasite populations and their relationships is necessary to study their implications for control intervention.     

Pf332-C231-reactive antibodies affect growth and development of intra-erythrocytic Plasmodium falciparum parasites

The Plasmodium falciparum antigen 332 (Pf332), is a megadalton parasite protein expressed at the surface of infected red cells during later stages of the parasite's developmental cycle. Antibodies to different parts of this antigen have been shown to inhibit parasite growth and adherence to host cells with or without ancillary cells. However, the mechanisms involved in these inhibitions remain largely unknown. We further analysed the activities of specific antibodies with regard to their specific mechanisms of action. For these analyses, affinity purified human antibodies against epitopes in the C-terminal fragment of Pf332 (Pf332-C231) were employed. All purified antibodies recognized Pf332-C231 both by immunofluorescence and ELISA. IgG was the main antibody isotype detected, although all sera investigated had varying proportions of IgG and IgM content. All the antibodies showed a capacity to inhibit parasite growth in P. falciparum cultures to different extents, mainly by acting on the more mature parasite stages. Morphological analysis revealed the antibody effects to be characterized by the presence of a high proportion of abnormal schizonts (15-30%) and pyknotic parasites. There was also an apparent antibody effect on the red cell integrity, as many developing parasites (up to 10% of trophozoites and schizonts) were extracellular. In some cases, the infected red cells appeared to be disintegrating/fading, staining paler than surrounding infected and uninfected cells. Antigen reversal of inhibition confirmed that these inhibitions were antigen specific. Furthermore, the growth of parasites after 22-42 h exposure to antibodies was investigated. Following the removal of antibody pressure, a decreased growth rate of these parasites was seen compared to that of control parasites. The present study confirms the potential of Pf332 as a target antigen for parasite neutralizing antibodies, and further indicates that epitopes within the C231 region of Pf332 should constitute important tools in the dissection of the role of Pf332 in the biology of the malaria parasite, as well as in the design of a malaria vaccine.     

Comparative morphometric analysis of bloodstream and lymph forms of Trypanosoma (T.) brucei brucei grown in vitro and in vivo

The fine structure of bloodstream forms of Trypanosoma brucei cultivated in vitro, and of trypanosomes from lymph and blood of mammalian hosts, was compared morphometrically. The cell volume, quantitative parameters of the mitochondrion and of glycosomes were mainly investigated. A Coulter Channelyzer was used for the first time to measure the mean cell volume of living parasites. In vitro, the monomorphic trypanosomes between the feeder layer cells showed lower values for mitochondrial parameters than the slightly pleomorphic forms from the supernatant medium. Trypanosomes in culture were very similar morphologically to forms from lymph nodes of rats. Despite some morphometric differences between cultivated blood stream forms and those grown in vivo, the similarity of both populations was clear. Both populations, however, differed significantly from stages found in the vector or from procyclic culture forms.     

Purification of mature schizonts of Plasmodium falciparum on colloidal silica gradients

The density of human erythrocytes infected in vitro with Plasmodium falciparum has been measured by isopycnic centrifugation in colloidal silica gradients. The densities of uninfected cells, rings, trophozoites, young schizonts, and mature schizonts were approximately 1.110, 1.110, 1.106, 1.097, and 1.090 g/ml, respectively. This information has been used to design a simple procedure for the separation of schizonts from other parasite stages and uninfected erythrocytes. By using synchronized cultures it is possible to obtain essentially pure schizonts after two centrifugations using a bench centrifuge. Such preparations are an excellent source of parasite antigen for immunological studies.     

The problem of cultivation of Mycobacterium leprae. A review with criteria for evaluating recent experimental work

Some criteria are presented to help evaluate papers appearing in the literature claiming successful cultivation of M. leprae either in the absence or in the presence of tissue-cultured cells. Recently, electron microscopic studies have definitely shown M. leprae to belong to the genus Mycobacterium and its division to occur through transverse section. A survey is given of the mycobacterial strains isolated in the last 10 years from leprosy lesions. These strains belong to taxonomically different species and cannot be considered to be M. leprae. No substantiated claim was made concerning the in vitro growth of M. leprae and the application of the tissue culture technique has been equally disappointing. The view is expressed that progress towards the in vitro cultivation of M. leprae can be made only as a result of increased knowledge about the intracellular environment and the metabolic activities of this organism, to be obtained by the application of modern biochemical and histochemical techniques.     

Mannosylated liposomes formulated with whole parasite P. falciparum blood-stage antigens are highly immunogenic in mice

The development of a blood-stage malaria vaccine has largely focused on the subunit approach. However, the limited success of this strategy, mainly due to antigenic polymorphism and the failure to maintain potent parasite-specific immune responses, indicates that other approaches must be considered. Whole parasite (WP) vaccines offer many advantages over sub-units; they represent every antigen on the organism, thus limiting the effects of antigenic polymorphism, and similarly they compensate for individual Immune-Response (Ir) gene-regulated non-responsiveness to any particular antigen. From a development perspective, they negate the need to identify and compare the relative efficacies of individual candidate antigens. WP vaccines induce protective immunity that is largely cell-mediated.However, WP blood-stage vaccines present a number of challenges for the development pathway. Key issues are cryopreservation and storage and the possible induction of antibodies against red blood cell surface antigens, even if the parasites are grown in blood group O, Rh negative blood. Here, we used a novel adaptation of an immunomagnetic method from STEMCELL™ Technologies to remove the red cell membranes from human red blood cells parasitized with P. falciparum. We then used these antigens to construct liposomes which were modified to present mannose on their membrane to target the liposome to antigen presenting cells. We then compared the immunogenicity of freshly prepared and lyophilized liposome vaccines. Following vaccination of mice, liposomes induced significantly lower antibody responses to human red cells but potent strain- and species-transcending cell-mediated immune responses to parasite antigens. These data support transitioning the P. falciparum liposomal vaccine into clinical studies.     

Use of the in vitro microtechnique for the assessment of drug sensitivity of Plasmodium falciparum in Sennar, Sudan

In 1978, studies on the chloroquine sensitivity of Plasmodium falciparum were carried out in the district of Sennar, Sudan. The results of the in vivo tests showed parasites resistant at the RI level only, but the mean clearance time of trophozoites from the blood was higher than for strains found in many other areas of tropical Africa. The in vitro tests, using the microtechnique, indicated a lower sensitivity to chloroquine in the local P. falciparum isolates than in those of most other African countries. However, similar results have been reported from Ethiopia. The chloroquine sensitivity of P. falciparum from Sennar is close to the critical level of resistance. The in vitro microtechnique was also used to test for the sensitivity to Dabequin, 4-aminobenzo-quinoline, and was generally found to be a suitable and reproducible method, with a greater potential than the standard macro method. At parasite densities of over 100 000 asexual parasites per microlitre of blood the effect of a given concentration of chloroquine was related to the parasite density owing to the selective uptake of the compound by the parasitized cells.     

The fine structure of Plasmodium falciparum and its host erythrocytes in natural malarial infections in man

Electron micrographs of ultra-thin sections of erythrocytes taken from two Liberian children ill with Plasmodium falciparum malaria show that the appliqué forms of this parasite are clearly within the host cell. The general fine structure of the parasites resembles that of other mammalian malaria parasites, as does the mode of ingestion of host cell material by pinocytosis. Granules of haemozoin were usually found in small vesicles pinched off from the large food vacuole. Scattered through the cytoplasm of infected red cells were narrow clear clefts bounded on each side by two unit membranes. These probably represent the Maurer''s clefts seen in light microscopy. The surface of infected erythrocytes was notably distorted, a phenomenon which may have a bearing on the stickiness of the infected red cells in human falciparum malaria and the segregation of these cells in the capillaries. Many uninfected erythrocytes showed a multiple alveolar, blister-like abnormality of a portion of the cell membrane; this was not seen in otherwise comparable blood from a case of P. ovale infection.     

Synthesis and in vitro antimalarial activity of tetraoxane-amine/amide conjugates

A series of tetraoxanes, tetraoxane-amine and tetraoxane-amide conjugates have been synthesized and screened for in vitro antimalarial activity against chloroquine-sensitive and chloroquine-resistant strains of Plasmodium falciparum. Most of the conjugates showed slightly better antimalarial activity than the parent tetraoxanes. Three of the conjugate compounds were potentially active with IC₅₀ values in the range of 0.38-0.80 μM. Cytotoxicity of four selected compounds was also evaluated in a panel of four cancer (SK-MEL, KB, BT-549, SK-OV-3) and two non-cancer (Vero and LLC-PK₁₁) cell lines up to a concentration of 25 μM and none of the compounds was found toxic to any of the cells.     

Limited genetic polymorphism of the Plasmodium vivax low molecular weight rhoptry protein complex in the Colombian population

Proteins involved in parasite adhesion and invasion are considered the best candidates for the development of asexual blood-stage antimalarial vaccines. Such vaccine candidates should be accessible by the immune system and have limited diversity. Considering the promising results obtained in previous trials by immunizing monkeys with the rhoptry-associated proteins 1 and 2 (RAP-1 and RAP-2), here we assessed the genetic variability of the Plasmodium vivax rap-1 and rap-2 genes isolated from Colombian parasite populations. Limited sequence diversity was found in these genes, possibly as a result of a functional/structural restriction. The presence of several haplotypes at relatively low frequencies and the excess of singleton mutations suggests that a demographic process might be affecting the loci. Our results support the inclusion of PvRAP-1 and PvRAP-2 in the design of an antimalarial subunit-based vaccine against P. vivax, which would avoid inducing allele-specific immunity.     

Diagnosis of Plasmodium falciparum infection in man: detection of parasite antigens by ELISA

An ELISA method has been developed for the diagnosis of Plasmodium falciparum infection in man. Parasites from in vitro cultures of P. falciparum were used as source of antigen for the solid phase and the source of specific antibody was immune Gambian sera; binding of antibody in antigen-coated wells was registered by means of alkaline phosphatase-conjugated anti-human IgG. Parasites were detected on the basis of inhibition of antibody-binding. The test was applied to the detection of parasites in human red blood cells (RBC) from in vitro cultures of P. falciparum and in RBC from infected Gambians; RBC from 100 Geneva blood donors served as normal, uninfected controls. In titration experiments, the degree of antibody-binding inhibition correlated with the number of parasites in the test RBC. Parasites were detected at a level of 8 parasites/10⁶ RBC. Samples of RBC were tested from 126 Gambians with microscopically proven infection; significant antibody-binding inhibition was found in 86% of these cases, where parasitaemia ranged from 10 to 125 000/μl of blood. The presence of high-titre antibody in the test preparations was found to reduce the sensitivity of parasite detection in infected RBC from in vitro cultures mixed with equal volumes of different antibody-containing sera. The sensitivity was restored in most cases by recovering the RBC by centrifugation before testing. In a preliminary experiment, there was no significant difference in antibody-binding inhibition using fresh infected RBC and RBC dried on filter-paper and recovered by elution, although there was greater variation in the latter samples.     

Induction of Multidrug Tolerance in Plasmodium falciparum by Extended Artemisinin Pressure

Plasmodium falciparum resistance to artemisinin derivatives in Southeast Asia threatens global malaria control strategies. Whether delayed parasite clearance, which exposes larger parasite numbers to artemisinins for longer times, selects higher-grade resistance remains unexplored. We investigated whether long-lasting artemisinin pressure selects a novel multidrug-tolerance profile. Although 50% inhibitory concentrations for 10 antimalarial drugs tested were unchanged, drug-tolerant parasites showed higher recrudescence rates for endoperoxides, quinolones, and an antifolate, including partner drugs of recommended combination therapies, but remained susceptible to atovaquone. Moreover, the age range of intraerythrocytic stages able to resist artemisinin was extended to older ring forms and trophozoites. Multidrug tolerance results from drug-induced quiescence, which enables parasites to survive exposure to unrelated antimalarial drugs that inhibit a variety of metabolic pathways. This novel resistance pattern should be urgently monitored in the field because this pattern is not detected by current assays and represents a major threat to antimalarial drug policy.