A chromosome 9 deletion in Plasmodium falciparum results in loss of cytoadherence

Many lines of Plasmodium falciparum undrgo a deletion of the right end of chromosome 9 during in vitro culture accompanied by loss of cytoadherence and gametocytogenesis. Selection of cytoadherent cells from a mixed population co-selects for those with an undeleted chromosome 9 and selected cells produce gametocytes. The deletion also results in loss of expression of PfEMP1, the putative cytoadherence ligand, suggesting PfEMP1 or a regulatory gene controlling PfEMP1 expression and gametocytogenesis may be encoded in this region. We have isolated several markers for the deleted region and are currently using a YAC-P. falciparum library to investigate this region of the genome in detail.

Characterization of a Plasmodium falciparum mutant that has deleted the majority of the gametocyte-specific Pf11-1 locus

We identified a gametocyte-specific protein of Plasmodium falciparum called Pf11-1 and provide experimental evidence that this molecule is involved in the emergence of gametes of the infected erythrocyte (gametogenesis). A mutant parasite clone, which has deleted over 90% of the PF11-1 gene locus, was an important control to establish the gametocyte-specific expression of the Pf11-1. Molecular analysis of the Pf11-1 deletion indicates that it is presumably due a chromosome breakage with subsequent "healing" by the addition of telomeric heptanucleotides. Moreover, similar DNA rearrangements are observed in most of the laboratory isolates during asexual propagation in vitro.

Immunogenicity and antigenicity of the N-term repeat amino acid sequence of the Plasmodium falciparum P126 antigen

The P126 protein, a parasitosphorus vacuole antigen of Plasmodium falciparum has beenshoen to induce protective immunity in Saimiri and Aotus monkeys. In the present work we investigated its immunogenicity. Our results suggest that the N-term of P126 is poorly immunogenic and antibody response against the P126 could be under a MHC restricted control in C57BL/6(H-2b) mice, which could be problematic in ternms of a use of the P126 in a vaccine program. However, we observed that a synthetic peptide, copying the 6 octapeptide repeat corresponding to the N-term of the P126, induces an antibody response to the native molecule in C57BL/6 non-responder mice. Moreover, the vaccine-P126 recombinant induced anmtibodies against the N-term of the molecule in rabbits while the unprocessed P126 did not.

The Pf332 gene codes for a megadalton protein of Plasmodium falciparum asexual blood stages

We characterized the Plasmodium falciparum antigen 332 (Ag332) which is specifically expressed during the asexual intraerythrocytic cycle of the parasite. The corresponding Pf332 gene has been located in the subtelomeric region of chromosome 11. Furthermore, it is present in all strais so far analyzed and shows marked restriction length fragment polymorphism. Partial sequence and restriction endonuclease digestion of cloned fragments revealed that the Pf332 gene is composed of highly degenerated repeats rich is glutamic acid. Mung been nuclease digestion and Northern blot analysis suggested that Pf332 gene codes for a protein of about 700 kDa. These data were further confirmed by Western blot and immunoprecipitation of parasites extracts with an antiserum raised against a recombinant clone expressing part of the Ag332. Confocal immunofluorescence showed that Ag332 is translocated from the parasite to the surface of infected red blood cells within vesicle-like structures. In addition, Ag332 was detected on the surface of monkey erythrocytes infected with Plasmodium falciparum.

Development of an immunoenzymatic assay using a monoclonal antibody against a 50-kDa catabolite from the P126 Plasmodium falciparum protein to the diagnosis of malaria infection

The WHO criterion of defering any donation of blood by a confirmed case of malaria for three years after cessation of therapy can not be applied in areas where malaria in endemic. For this reason we developed an immunoenzymatic assay for the detection of plasmodial antigens for blood screening in malararial endemic areas. So, we tested sera from 191 individuals. Among patients with active disease 100% of the cases of Plasmodium falciparum or mixed infections and 91.7% of those with P. vivax were positive for the presence of plasmodial antigens. The lower parasitaemia detected was 0.0003% for P. vivax malária. When the frequency of positive circulating malarial antigens was evaluated among asymptomatic and symptomatic individuals with negative TBS, positive results were found in respectively 38.7% and 17.7% of the individuals studied in the 30 days after confirmed malaria attack. Data provide by these assays have shown that ELISA seemed to be more sensitive than parasitological examination for malaria diagnosis. This test by virtue of its high sensivity and the facilities in processing a large number of specimens, can prove to be useful in endemic areas for the recognition of asymptomatic malaria and screening of blood donors.

T cell responses to repeat and non-repeat regions of the circumsporozoite protein detected in volunteers immunized with Plasmodium falciparum sporozoites

The design of malarial vaccine based on the circumsporozoite (CS) protein, a majuor surface antigen of the sporozoite stage of the malaria parasite, requires the identification of T and B cell epitopes for inclusion in recombinant or synthetic vaccine candidates. We have investigated the specificity and function of a series of T cell clones, derived from volunteers immunized with Plasmodium falciparum sporozoites in an effort to identify relevant epitopes in the immune response to the pre-erythrocytic stages of the parasite. CD4+ T cell clones were obtained wich specifically recognized a repetitive epitope located in the 5'repeat region of the CS protein. This epitope, when conjugated to the 3'repeat region in a synthetic MAPs construct, induced high titers of antisporozoite antibodies in C57B1 mice. A second T cell epitope, which mapped to aa 326-345 of the carboxy terminal, was recognized by lytic, as well as non-lytic, CD4+ T cells derived from the sporozoite-immunized volunteers. The demonstration of CD4+ CTL in the volunteers, and the recent studies inthe rodent model (Renia et al., 1991; Tsuji et al., 1990), suggested that CS-specific CD4+ T cells, in addition to their indirect role as helper cells in the induction of antibody and CD8 + effector cells, may also play a direct role in protection against sporozoite challenge by targeting EEF within the liver.

Plasmodium falciparum CS protein - prime malaria vaccine candidate: definition of the human CTL domain and analysis of its variation

Studies in mice have shown that immunity to malaria sporozoites is mediated primarily by citotoxic T lymphocytes (CTL) specific for epitopes within the circumsporozoite (CS) protein. Humans, had never been shown to generate CTL against any malaria or other parasite protein. The design of a sub-unit vaccine for humans ralies on the epitopes recognized by CTL being identified and polymorphisms therein being defined. We have developed a novel technique using an entire series of overlapping synthetic peptides to define the epitopes of the Plasmodium falciparum CS protein recognized by human CTL and have analyzed the sequence variation of the protein with respect to the identified CTL epitopic domain. We have demonstrated that some humans can indeed generate CTL. against the P. falciparum CS protein. Furthermore, the extent of variation observed for the CTL recognition domain is finite and the combination of peptides necessary for inclusion in a polyvalent vaccine may be small. If ways can be found to increase immune responsiveness, then a vaccine designed to stimulate CS protein-specific CTL activity may prevent malaria.

Optimization and inhibition of the adherent ability of Plasmodium falciparum-infected erythrocytes

The vast majority of the 1-2 million malaria associated deaths that occur each year are due to anemia and cerebral malaria (the attachment of erythrocytes containing mature forms of Plasmodium falciparum to the endothelial cells that line the vascular beds of the brain). A "model" system"for the study of cerebral malaria employs amelanotic melanoma cells as the "target"cells in an vitro cytoadherence assay. Using this model system we determined that the optimum pH for adherence is 6.6 to 6.8, that high concentrations of Ca²* (50mM) result in increased levels of binding, and that the type of buffer used influences adherence (Bis Tris > MOPS > HEPES > PIPES). We also observed that the ability of infected erythrocytes to cytoadhere varied from (erythrocyte) donor to donor. We have produced murine monoclonal antibodies against P. falciparum-infected red cells which recognized modified forms of human band 3; these inhibit the adherence of infected erythrocytes to melanoma cells in a doso responsive fashion. Antimalarials (chloroquine, quinacrine, mefloquine, artemisinin), on the other hand, affected adherence in an indirect fashion i.e. since cytoadherence is due, in part to the presence of knobs on the surface of the infected erythrocyte, and knob formation is dependent on intracellular parasite growth, when plasmodial development is inhibited so is knob production, and consequently adherence is ablated.

Molecular mechanisms and biological importance of Plasmodium falciparum erythrocyte rosetting

Rosetting, i.e. the spontaneous binding of uninfected to malaria infected erythrocytes and endothelial cytoadherence may hinder the blood flow and lead to serve Plasmodium falciparum malaria. Falciparum isolates obtained from unconscious patients all form rosettes and/or express a significantly higher man rosetting rate than isolates from patients with uncomplicated malaria. Furthermore, sera of patients with cerebral malaria are devoid of anti-rosetting activity while sera from patients with mild disease carry high levels of anti-rosetting antibodies. The presence of anti-rosetting antibodies also seems important for the efficient interaction of rosetting infected rbc and leucocytes. Two parasite derived rosetting ligands of Mr 22k and Mr28K named "rosettins, have been found on the surface of rosetting infected erythrocytes. CD36 has in at least some strains of parasites been found to function as a rosetting receptor on the uninfectederythrocyte. Heparin disrupts rosettes of P. falciparum in vitro and inhibits the sequestration of rosetting cells ex vivo. In conclusion, rosetting seems a crucial factor in the development of cerebral malaria and treatment of patients with anti-rosetting substances might become an effectivew adjunct in the treatment of severe malaria.

Epidemiological distribution of Plasmodium falciparum drug resistance in Brazil and its relevance to the treatment and control of malaria

With the use of a simple formulary, field by health agents was ewstablished a monitoring programme for responses of P. falciparum to the antimalarial drugs. This monitoring programme is emphasized for knowledge of the epidemiology of the drug resistance and the control of malaria falciparum in Amazonan Basin where occurs more than 95% of Brazilian malaria cases every year. It was demonstrated that still now 4-aminoquinolines have a great importance for the mortality control in areas where just SUCAM (National Health Foundation - Health Ministry) agenst are present without any medical assistance. The results obtained permitted the simplification of malaria treatment in Brazil Important conclusions were established in the field of malaria drug resistance.