Expression of Plasmodium falciparum surface antigens in Escherichia coli.

The asexual blood stages of the human malarial parasite Plasmodium falciparum produce many antigens, only some of which are important for protective immunity. Most of the putative protective antigens are believed to be expressed in schizonts and merozoites, the late stages of the asexual cycle. With the aim of cloning and characterizing genes for important parasite antigens, we used late-stage P. falciparum mRNA to construct a library of cDNA sequences inserted in the Escherichia coli expression vector pUC8. Nine thousand clones from the expression library were immunologically screened in situ with serum from Aotus monkeys immune to P. falciparum, and 95 clones expressing parasite antigens were identified. Mice were immunized with lysates from 49 of the bacterial clones that reacted with Aotus sera, and the mouse sera were tested for their reactivity with parasite antigens by indirect immunofluorescence, immunoprecipitation, and immunoblotting assays. Several different P. falciparum antigens were identified by these assays. Indirect immunofluorescence studies of extracellular merozoites showed that three of these antigens appear to be located on the merozoite surface. Thus, we have identified cDNA clones to three different P. falciparum antigens that may be important in protective immunity.     

Uninfected erythrocytes form "rosettes" around Plasmodium falciparum infected erythrocytes

The human malaria parasite, P. falciparum, exhibits cytoadherence properties whereby infected erythrocytes containing mature parasite stages bind to endothelial cells both in vivo and in vitro. Another property of cytoadherence, "rosetting," or the binding of uninfected erythrocytes around an infected erythrocyte, has been demonstrated with a simian malaria parasite P. fragile which is sequestered in vivo in its natural host, Macaca sinica. In the present study we demonstrate that rosetting occurs in P. falciparum. Rosetting in P. falciparum is abolished by protease treatment and reappears on further parasite growth indicating that, as in P. fragile, it is mediated by parasite induced molecules which are protein in nature. P. vivax and P. cynomolgi, which are not sequestered in the host, did not exhibit rosetting. Rosetting thus appears to be a specific property of cytoadherence in malaria parasites.     

Gene selective mRNA cleavage inhibits the development of Plasmodium falciparum

Unique peptide-morpholino oligomer (PMO) conjugates have been designed to bind and promote the cleavage of specific mRNA as a tool to inhibit gene function and parasite growth. The new conjugates were validated using the P. falciparum gyrase mRNA as a target (PfGyrA). Assays in vitro demonstrated a selective degradation of the PfGyrA mRNA directed by the external guide sequences, which are morpholino oligomers in the conjugates. Fluorescence microscopy revealed that labeled conjugates are delivered into Plasmodium-infected erythrocytes during all intraerythrocytic stages of parasite development. Consistent with the expression of PfGyrA in all stages of parasite development, proliferation assays showed that these conjugates have potent antimalarial activity, blocking early development, maturation, and replication of the parasite. The conjugates were equally effective against drug sensitive and resistant P. falciparum strains. The potency, selectivity, and predicted safety of PMO conjugates make this approach attractive for the development of a unique class of target-specific antimalarials and for large-scale functional analysis of the malarial genome.     

Molecular analysis of chloroquine resistance in Plasmodium falciparum in Yunnan Province, China

Resistance of Plasmodium falciparum to chloroquine (CQ) is determined by the mutation at K76T of the P. falciparum chloroquine resistance transporter (pfcrt) gene and modified by other mutations in this gene and in the P. falciparum multidrug resistance 1 (pfmdr1) gene. To determine the extent of polymorphisms in these genes in field P. falciparum isolates from Yunnan province of China, we genotyped the pfcrt codon 76, pfmdr1 codons 86 and 1246. Our results showed that although CQ has been withdrawn from treating falciparum malaria for over two decades, 90.3% of the parasites still carried the pfcrt K76T mutation. In contrast, mutations at pfmdr1 codons 86 and 1246 were rare. Sequencing analysis of the pfcrt gene in 34 parasite field isolates revealed CVIET at positions 72-76 as the major type, consistent with the theory of Southeast Asian origin of CQ resistance in the parasite. In addition, two novel pfcrt haplotypes (75D/144Y/220A and 75E/144Y/220A) were identified. Real-time polymerase chain reaction was used to determine pfmdr1 gene amplification, which is associated with mefloquine resistance. Our result indicated that in agreement with that mefloquine has not been used in this area, most (>90%) of the parasites had one pfmdr1 copy. Genotyping at two hypervariable loci showed relatively low levels of genetic diversity of the parasite population. Meanwhile, 28.4% of cases were found to contain mixed clones, which favour genetic recombination. Furthermore, despite a unique history of antimalarial drugs in Yunnan, its geographical connections with three malarious countries facilitate gene flow among parasite populations and evolution of novel drug-resistant genotypes. Therefore, continuous surveillance of drug resistance in this area is necessary for timely adjustment of local drug policies and more effective malaria control.     

New emerging Plasmodium falciparum genotypes in children during the transition phase from asymptomatic parasitemia to malaria

Semi-immunity against Plasmodium falciparum occurs after many infections. In areas of high malaria transmission, the prevalence of asymptomatic parasite carriers increases with age. We investigated P. falciparum genotypes in a cohort of asymptomatic carriers who were followed until they became symptomatic. Blood spots on filter paper and blood smears were collected daily from 10 children in Lambaréné, Gabon. The parasite genotypes present on successive days were determined by a polymerase chain reaction using the polymorphic region of the merozoite surface antigen-2 for typing. The same parasite genotypes persisted in eight out of ten children and parasite densities were low throughout the asymptomatic phase indicating inhibition of parasite growth. Appearance of symptoms was associated with an increase in parasitemia and appearance of novel parasite genotypes. The results suggest that the parasites causing a clinical episode are those against which a child has not yet mounted an efficient protective immune response.     

An iron-carboxylate bond links the heme units of malaria pigment.

The intraerythrocytic malaria parasite uses hemoglobin as a major nutrient source. Digestion of hemoglobin releases heme, which the parasite converts into an insoluble microcrystalline material called hemozoin or malaria pigment. We have purified hemozoin from the human malaria organism Plasmodium falciparum and have used infrared spectroscopy, x-ray absorption spectroscopy, and chemical synthesis to determine its structure. The molecule consists of an unusual polymer of hemes linked between the central ferric ion of one heme and a carboxylate side-group oxygen of another. The hemes are sequestered via this linkage into an insoluble product, providing a unique way for the malaria parasite to avoid the toxicity associated with soluble heme.     

泛种特异性抗疟原虫单克隆抗体M26-32的分子生物学特性研究 Ⅱ.靶抗原基因的筛选和序列分析

用泛种特异性抗疟原虫单克隆抗体Ｍ２６－３２作探针，筛选恶性疟原虫ｃＤＮＡ基因表达文库。结果获得一段含ＮＫＮＤ４个氨基酸靶抗原决定簇表位基因的４７２个碱基的基因序列。用该基因序列作探针与不同种的ＤＮＡ进行杂交，证实该基因序列为一段疟原虫基因序列。对该基因片段在染色体内的定位分析发现，该基因序列定位于恶性疟原虫的第７对染色体上，且存在于多种不同分离株（包括抗氯喹株）的恶性疟原虫的ＤＮＡ之中。通过ＧｅｎｅＢａｎｋ检索证实，该基因序列与已知的其它疟原虫基因序列不同，是一段新发现的，存在于不同种株疟原虫基因的单克隆抗体Ｍ２６－３２的靶抗原基因序列片段。     

A reappraisal of the effects of iron and desferrioxamine on the growth of Plasmodium falciparum''in vitro'': the unimportance of serum iron

It has been suggested that P. falciparum takes up iron from serum and that desferrioxamine, an iron chelating agent, inhibits parasite growth. We have now shown, however, that when all the iron is transferrin bound, P. falciparum, in culture, takes up less than 7 pmol Fe/10(9) parasites/24 h and that incorporation is increased only in the presence of a high molecular weight iron complex not naturally found in serum. Furthermore, removal of iron serum did not reduce parasite growth, and addition of excess iron was inhibitory. Desferrioxamine inhibited growth, but this inhibition was reduced under conditions in which the transfer of iron from transferrin to desferrioxamine was accelerated. We conclude that P. falciparum does not directly utilize serum iron and that desferrioxamine does not inhibit the parasite by interfering with the supply of iron from the incubation medium. The results are relevant to clinical data which suggest that added nutritional iron enhances the host susceptibility to malaria.     

Genes necessary for expression of a virulence determinant and for transmission of Plasmodium falciparum are located on a 0.3-megabase region of chromosome 9.

Virulence of the human malaria parasite Plasmodium falciparum is believed to relate to adhesion of parasitized erythrocytes to postcapillary venular endothelium (asexual cytoadherence). Transmission of malaria to the mosquito vector involves a switch from asexual to sexual development (gametocytogenesis). Continuous in vitro culture of P. falciparum frequently results in irreversible loss of asexual cytoadherence and gametocytogenesis. Field isolates and cloned lines differing in expression of these phenotypes were karyotyped by pulse-field gel electrophoresis. This analysis showed that expression of both phenotypes mapped to a 0.3-Mb subtelomeric deletion of chromosome 9. This deletion frequently occurs during adaptation of parasite isolates to in vitro culture. Parasites with this deletion did not express the variant surface agglutination phenotype and the putative asexual cytoadherence ligand designated P. falciparum erythrocyte membrane protein 1, which has recently been shown to undergo antigenic variation. The syntenic relationship between asexual cytoadherence and gametocytogenesis suggests that expression of these phenotypes is genetically linked. One explanation for this linkage is that both developmental pathways share a common cytoadherence mechanism. This proposed biological and genetic linkage between a virulence factor (asexual cytoadherence) and transmissibility (gametocytogenesis) would help explain why a high degree of virulence has evolved and been maintained in falciparum malaria.     

Identification and partial characterization of exoantigens derived from medium used to culture Plasmodium falciparum

Rabbits were immunized with exoantigens from the spent medium of Plasmodium falciparum cultures, and the resultant immunologic responses were studied by indirect fluorescent antibody (IFA), hemagglutination (HA), and two-dimensional crossed immunoelectrophoretic (IEP) techniques. By crossed IEP, three parasite antigens, identified and characterized as proteins lacking lipid and carbohydrate moieties, reacted with rabbit antiserum and human immune serum. Pre-immunization sera of the rabbits used in these experiments had 1:80 IFA titers against P. falciparum schizonts which were then boosted eightfold by immunization with parasite exoantigens, in contrast to IFA titers of less than 1:2 for coccidia- and Pasteurella-free rabbits. Experimental infections of coccidia- and Pasteurella-free rabbits with rabbit Eimeria spp. resulted in 1:80 anti-P. falciparum IFA titers, suggesting cross-reactivity of coccidial and plasmodial antigens. Post-immunization sera demonstrated extremely high HA titers against human erythrocytes, underscoring the potency of human blood components containing parasite antigens prepared from cultures. These results suggest that coccidia- and Pasteurella-free rabbits may be useful in the analysis of antigen of cultured P. falciparum.     

Periodic and chaotic host-parasite interactions in human malaria.

It has been recognized since ancient times that malaria fever is highly periodic but the mechanism has been poorly understood. Malaria fever is related to the parasite growth cycle in erythrocytes. After a fixed period of replication, a mature parasite (schizont) causes the infected erythrocyte to rupture, releasing progeny that quickly invade other erythrocytes. Simultaneous rupture of a large number of schizonts stimulates a host fever response. Febrile temperatures are damaging to Plasmodium falciparum, particularly in the second half of its 48-hr replicative cycle. Using a mathematical model, we show that these interactions naturally tend to generate periodic fever. The model predicts chaotic parasite population dynamics at high multiplication rates, consistent with the classical observation that P. falciparum causes less regular fever than other species of parasite.     

Antigenic variation in Plasmodium falciparum.

Antigenic variation of infectious organisms is a major factor in evasion of the host immune response. However, there has been no definitive demonstration of this phenomenon in the malaria parasite Plasmodium falciparum. In this study, cloned parasites were examined serologically and biochemically for the expression of erythrocyte surface antigens. A cloned line of P. falciparum gave rise to progeny that expressed antigenically distinct forms of an erythrocyte surface antigen but were otherwise identical. This demonstrates that antigenic differences on the surface of P. falciparum-infected erythrocytes can arise by antigenic variation of clonal parasite populations. The antigenic differences were shown to result from antigenic variation of the parasite-encoded protein, the P. falciparum erythrocyte membrane protein 1.     

Tetracycline analogue-regulated transgene expression in Plasmodium falciparum blood stages using Toxoplasma gondii transactivators

Genetic manipulation has revolutionized research in the Apicomplexan parasite Plasmodium falciparum, the most important causative agent of malaria. However, to date no techniques have been established that allow modifications that are deleterious to blood-stage growth, such as the disruption of essential genes or the expression of dominant-negative transgenes. The recent establishment of a screen for functional transactivators in the related parasite Toxoplasma gondii prompted us to identify transactivators in T. gondii and to examine their functionality in P. falciparum. Tetracycline-responsive minimal promoters were generated based on the characterized P. falciparum calmodulin promoter and used to assess transactivators in P. falciparum. We demonstrate that artificial tetracycline-regulated transactivators isolated in T. gondii are also functional in P. falciparum. By using the tetracycline analogue anhydrotetracycline, efficient, stage-specific gene regulation was achieved in P. falciparum. This regulatable expression technology has clear potential for the study of essential gene function in P. falciparum blood stages. On the other hand, the identified transactivators are not functional in mammalian cells, consistent with the fundamental differences in the mechanism of gene regulation between Apicomplexan parasites and their human hosts.     

Potential of the Panama strain of Plasmodium vivax for the testing of malarial vaccines in Aotus nancymai monkeys

Aotus monkeys were infected with a strain of Plasmodium vivax from Panama to determine its potential for the testing of malarial vaccines. After sporozoite inoculation, 3 splenectomized Aotus nancymai that had been infected previously with Plasmodium falciparum and P. vivax had prepatent periods of 13, 15, and 15 days with maximum parasite counts of 12,726/microl, 5,310/microl, and 9,180/microl. Three other A. nancymai previously infected with P. falciparum only had prepatent periods of 17, 15, and 15 days with maximum parasite counts of 44,460/microl, 31,500/microl, and 42,660/microl. One monkey with no previous history of infection had a prepatent period of 14 days after sporozoite inoculation, and a maximum parasite count of 100,000/microl; detectable parasitemia persisted for almost 500 days with 13 recognizable peaks in the parasite count. Anopheles dirus, Anopheles freeborni, Anopheles stephensi, and Anopheles quadrimaculatus mosquitoes were readily infected with the Panama strain.     

Multiplex PCR assay for the detection of Anopheles fluviatilis species complex, human host preference, and Plasmodium falciparum sporozoite presence, using a unique mosquito processing method

A multiplex PCR assay has been developed for detection of Anopheles fluviatilis cryptic species, their human host preference, and Plasmodium falciparum presence in the mosquito. PCR conditions were optimized using primer sets specific for A. fluviatilis cryptic species, Homo sapiens, and P. falciparum and evaluated with field-collected mosquitoes. A unique mosquito processing method was used for screening P. falciparum carrying capacity and human host preference of A. fluviatilis mosquitoes in first-round multiplex PCR. The vectorial status of the mosquito for P. falciparum parasite was confirmed in second-round PCR. Of the 121 collected mosquitoes, 92 were of S type, 26 of T type, and 3 were of other types. Human host preference was dominant in S type, of which 4% were P. falciparum sporozoite positive. This assay and processing method can also be used to evaluate vector competence of other anophelines.     

A longitudinal study of P. falciparum response to chloroquine in relation to the epidemiology

An attempt has been made to study the dynamics in the epidemiology of a locality where the local strains of Plasmodium falciparum have developed resistance to the antimalarial chloroquine. Increase in the transmission rate and prolonged drug pressure with chloroquine have been noted in association with the development of resistance. The importance of the study is on the mean parasite clearance time and mean parasite recrudescence time which appear to be related with the process developing resistance of chloroquine.