IgE elevation and IgE anti-malarial antibodies in Plasmodium falciparum malaria: association of high IgE levels with cerebral malaria.

In the course of studying immunoregulation in human Plasmodium falciparum malaria we have investigated IgE levels and IgE anti-plasmodial antibodies in children and adults from areas of high malaria endemicity in both Africa and Asia. On average, 85% of all donors had significantly elevated levels of total IgE. A fraction of the IgE had anti-plasmodial activity as revealed by ELISA with lysates of infected erythrocytes as antigen. Using synthetic peptides representing antigenic regions of two major plasmodial blood stage antigens, IgE antibody concentrations ranged from 5 to 15 ng/ml serum for each of the peptides. On average, the concentrations of the corresponding IgG antibodies were x 500-1000 higher. Immunoblotting of parasite lysates showed that most donors had IgE antibodies against one or several of a restricted number of plasmodial polypeptides, with antibodies against an antigen of mol.wt 45 kD already being present in all donors at an early age. Donors having IgE antibodies to particular antigens also frequently had corresponding IgG4 antibodies, reflecting underlying IL-4-dependent cellular mechanisms controlling formation of these isotypes. As infection with other parasites such as helminths is known to induce IgE elevation, the results do not prove that plasmodial infections were the primary cause of IgE induction. However, the importance of plasmodial infection for IgE elevation was supported by the finding of significantly higher levels of IgE, but not of IgG, in children with cerebral malaria compared with patients with uncomplicated disease.     

Antibodies to the N-terminal block 2 of Plasmodium falciparum merozoite surface protein 1 are associated with protection against clinical malaria

This longitudinal prospective study shows that antibodies to the N-terminal block 2 region of the Plasmodium falciparum merozoite surface protein 1 (MSP-1) are associated with protection against clinical malaria in an area of stable but seasonal malaria transmission of Ghana. Antibodies to the block 2 region of MSP-1 were measured in a cohort of 280 children before the beginning of the major malaria transmission season. The cohort was then actively monitored for malaria, clinically and parasitologically, over a period of 17 months. Evidence is presented for an association between antibody responses to block 2 and a significantly reduced risk of subsequent clinical malaria. Furthermore, statistical survival analysis provides new information on the duration of the effect over time. The results support a conclusion that the block 2 region of MSP-1 is a target of protective immunity against P. falciparum and, thus, a promising new candidate for the development of a malaria vaccine.     

Antibodies to the ring-infected erythrocyte surface antigen of Plasmodium falciparum elicited by infection with Plasmodium malariae.

The ring-infected erythrocyte surface antigen (RESA) of Plasmodium falciparum (RESA-P), found in the membrane of erythrocytes infected with young asexual stages of P. falciparum, is a promising vaccine candidate. Antibodies to RESA-P were inducible by infection with another human malaria species, P. malariae. Of 298 serum samples from inhabitants of three isolated localities in Peru where P. vivax and P. malariae were endemic and P. falciparum had never been reported, 26% had anti-RESA-P antibodies as evidenced by a modified immunofluorescent-antibody assay and confirmed by Western blot (immunoblot) analysis. These seroepidemiologic observations were corroborated by the fact that of six chimpanzees infected with P. malariae, three developed anti-RESA-P antibodies after infection. The modified immunofluorescent-antibody-reactive antibodies, purified by adsorption and elution on monolayers of glutaraldehyde-fixed and air-dried P. falciparum-infected erythrocytes, reacted in an immunofluorescent-antibody assay with both parasite structures and erythrocyte membrane in P. falciparum antigen preparations, but only with parasite structures in P. malariae antigen preparations. This serologic cross-reactivity between P. falciparum and P. malariae is of interest in view of the importance of RESA-P as a vaccine candidate and because the two species are coendemic in many areas.     

Antibodies that inhibit Plasmodium falciparum adhesion to chondroitin sulfate A are associated with increased birth weight and the gestational age of newborns

Antibodies that inhibit Plasmodium falciparum adhesion to the placental receptor chondroitin sulfate A are associated with a reduced risk of placental malaria, but whether these antibodies lead to improved pregnancy outcomes is unknown. We measured antiadhesion antibody levels in parturient women in western Kenya, where malaria transmission is intense. Secundigravid women with antiadhesion activity in their plasma delivered babies that were on average 398 g heavier (P = 0.019) and 2 weeks more mature (P = 0.002) than babies delivered to secundigravidas without antiadhesion activity. Our findings support the development of antiadhesion vaccines to prevent poor fetal outcomes due to pregnancy malaria.     

Inhibition of the growth of Plasmodium falciparum and Plasmodium berghei by the DNA polymerase inhibitor HPMPA.

The acyclic adenosine analogue (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine [HPMPA] belongs to a class of nucleoside analogues originally described as having potent activity against a broad spectrum of DNA viruses. We examined the effects of this class of drugs on the growth of cultured Plasmodium falciparum. Strong inhibition was observed by HPMPA (ID50 = 47 nM) at concentrations more than 1000-fold less than the cytotoxic dose for human cells. 3-deaza-HPMPA was even more strongly inhibitory (ID50 = 8 nM), whereas several other acyclic nucleosides were not effective. In mice infected with Plasmodium berghei, increase of parasitaemia can be blocked for 4-6 days by a single injection of HPMPA. Repeated drug administration blocks parasite growth for prolonged periods at doses that are clinically feasible. We also determined the inhibition of several purified Plasmodium DNA polymerases by diphosphorylated HPMPA (HPMPApp). DNA polymerase alpha-like enzymes of P. falciparum and P. berghei are inhibited with an IC50 = 40 microM and a gamma-like DNA polymerase from P. falciparum is even 40-fold more sensitive to the drug. The inhibition by HPMPApp is competitive with dATP, strongly suggesting that Plasmodium DNA polymerases are targets for this class of nucleotide analogue.     

Inhibition of the in vitro growth of Plasmodium falciparum by D vitamins and vitamin D-3 derivatives

D vitamins are effective inhibitors of the in vitro intraerythrocytic growth of Plasmodium falciparum. Disappearance of the parasitemia was observed after 48 h contact between infected cells and 5 x 10(-6) M 1 alpha-hydroxycholecalciferol, 5 x 10(-5) M 25-hydroxycholecalciferol (25-OH-D-3), 1 alpha, 25-dihydroxycholecalciferol or 2.5 x 10(-4) vitamin D-2 and D-3. A 48 h pretreatment of healthy erythrocytes with 5 x 10(-5) M 25-OH-D-3 did not change their susceptibility to invasion by the parasite and their ability to support the growth of P. falciparum. Ionomycin, a calcium ionophore, and EGTA prevented parasite development at concentrations greater than 2 x 10(-7) M and 4 x 10(-4) M, respectively, but did not antagonize the inhibitory activity of 25-OH-D-3. Addition of 25-OH-D-3 for 12 or 24 h duration to synchronized cultures, showed that the drug had a schizonticidal action, but was without effect when parasites were in the ring form.     

Baculovirus-mediated expression of Plasmodium falciparum erythrocyte binding antigen 175 polypeptides and their recognition by human antibodies.

The erythrocyte binding antigen EBA-175 is a 175-kDa Plasmodium falciparum protein which mediates merozoite invasion of erythrocytes in a sialic acid-dependent manner. The purpose of this study was to produce recombinant EBA-175 polypeptide domains which have previously been identified as being involved in the interaction of EBA-175 with erythrocytes and to determine whether these polypeptides are recognized by malaria-specific antibodies. The eba-175 gene was cloned by PCR from genomic DNA isolated from the 3D7 strain of P. falciparum. The predicted protein sequence was highly conserved with that predicted from the published eba-175 gene sequences from the Camp and FCR-3 strains of P. falciparum and contained the F segment divergent region. Purified recombinant EBA-175 polypeptide fragments, expressed as glutathione S-transferase fusion proteins in insect cells by using the baculovirus system, were recognized by antibodies present in serum from a drug-cured, malaria-immune Aotus nancymai monkey. The fusion proteins were also recognized by antibodies present in sera from individuals residing in areas where malaria is endemic. In both cases the antibodies specifically recognized the EBA-175 polypeptide portion of the fusion proteins. Antibodies raised in rabbits immunized with the recombinant fusion proteins recognized parasite proteins present in schizont-infected erythrocytes. Our results suggest that these regions of the EBA-175 protein are targets for the immune response against malaria and support their further study as possible vaccine components.     

Correlation of the efficiency of fatty acid derivatives in suppressing Plasmodium falciparum growth in culture with their inhibitory effect on acyl-CoA synthetase activity

The intraerythrocytic malaria parasite depends on the surrounding medium for a supply of phospholipid precursors. Efficient inhibition (IC50 7-90 microM) of Plasmodium falciparum growth in vitro was achieved using modified fatty acids. The fatty acid analogues most effective in suppressing P. falciparum growth in culture were also the most active inhibitors of acyl-CoA synthetase from the monkey parasite P. knowlesi.     

The toxic actions of MPTP and its metabolite MPP+ are not mimicked by analogues of MPTP lacking an N-methyl moiety

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), its metabolite 1-methyl-4-phenylpyridine (MPP+) and three analogues of MPTP, lacking an N-methyl moiety, namely, 4-phenylpiperidine (I), 4-phenyl-1,2,3,6-tetrahydropyridine (II) and 4-phenylpyridine (III), were infused continuously for a period of 4 days into the rat substantia nigra. Within 12 h of commencing the bilateral infusion of MPTP or MPP+, rats showed marked motor deficits with reduction in locomotor activity, loss of ability to move the forelimbs and grip with forepaws and, following MPP+ infusions, similar loss of movement in the hindlimbs associated with the development of limb and body rigidity. These motor deficits were not induced by the 3 analogues of MPTP on infusion into the substantia nigra. After 4 days of infusion, the motor deficits caused by MPTP and, in particular, MPP+, were still marked, and for MPP+ these correlated with marked loss of striatal dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid. 4-Phenyl-1,2,3,6-tetrahydropyridine caused a small loss in striatal DA and DOPAC, but the other analogues failed to modify the striatal content of DA or its metabolites. Small alterations of chemical structures related to MPTP and its metabolite can critically alter ability to induce behavioural and neurochemical changes reflecting toxicity on the nigrostriatal DA system.     

Molecules on the surface of the Plasmodium falciparum infected erythrocyte and their role in malaria pathogenesis and immune evasion.

The surface of the erythrocyte undergoes a number of modifications during infection by Plasmodium falciparum. These modifications are critical for pathogenesis of severe disease and the acquisition of host immunity through their role in interactions between the host and the parasite and in antigenic variation. Our knowledge of the molecular basis for these processes has increased dramatically over the last few years, through a combination of genomic and biochemical studies. This review provides a summary of the molecules involved in cytoadherence and antigenic variation in P. falciparum.     

Detection of antigens and antibodies in the urine of humans with Plasmodium falciparum malaria.

Humans infected with Plasmodium falciparum frequently have elevated levels of proteins in their urine, but it is unclear if any of these proteins are parasite antigens or antimalarial antibodies. To resolve this question, urine samples from malaria patients and controls living in Thailand and Ghana were evaluated. Urine samples from 85% of the patients had elevated protein levels and contained proteins with Mrs ranging from less than 29,000 to greater than 224,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antisera were produced against urine from infected and control subjects. Antisera raised against infected, but not control, urine were positive by indirect immunofluorescence on P. falciparum parasites and immunoprecipitated approximately 12 unique bands from extracts of parasites metabolically labeled with 35S-methionine. These data suggest that a variety of P. falciparum antigens are released into urine during acute infection. It is also likely that anti-P. falciparum antibodies are present in the urine of malaria patients because samples from these patients, but not controls, were positive in indirect immunofluorescence assays and immunoprecipitated at least 19 P. falciparum antigens from extracts of metabolically labeled parasites. The detection of malarial antigens and antibodies in urine may lead to a new approach for the diagnosis of malaria.     

Plasmodium falciparum chloroquine and pyrimethamine resistance monitoring network with molecular tools in the Niger River valley, Republic of Niger

Plasmodium falciparum resistance to chloroquine first arose in Africa 25 years ago. Nowadays most of African malaria control programmes have switched their first-line treatment of uncomplicated malaria cases towards artemisinin derivatives combination. After WHO guidelines, a survey network for malaria treatment resistance has been set up in the Niger valley around Niamey since December 2004. The association of the Niger national malaria control programme with the CERMES research center allowed collecting of samples from both health centers and hospitals of this region. Blood finger-pricks on filter papers were tested for detection of plasmodial antigen in health center without biological diagnosis capacity. Specimens found positive either in hospital laboratory or by using antigen method were tested by PCR/RFLP to detect K76T mutations on the pfcrt gene and S108N mutation on the pfdhfr gene. This simple procedure allows the screening of a large number of specimens. Moreover, a spatial distribution of mutations and evidence of resistance clusters were searched integrating the data in a geographic information system. The 76T mutation of pfcrt and 108N of pfdhfr were respectively found in 50.8% and 57% of the specimens tested. No statistically significant difference was found according to the level of sanitary formations or the age of the patients. No resistance cluster was identified and the prevalence of mutation seems homogeneous in the zone. By completing the clinical efficacy studies we think that our simple method for collecting and testing blood samples associated with clinical efficacy studies may be useful for building a network of malaria drug resistance in Africa.     

Detection of a LFA-1-like epitope on the surface of erythrocytes infected with a strain of Plasmodium falciparum.

The adhesion of erythrocytes infected with Plasmodium falciparum (P. falciparum) is one of the major pathological features of severe malaria. Several potential receptors to endothelium for falciparum-infected erythrocyte on endothelium have been described. Recently, the malaria binding site on ICAM-1(CD54) has been mapped to a site distinct but overlapping with the LFA-1 (CD11a/CD18) site. We detected by flow cytometry, confocal laser microscopy and immunoprecipitation, a molecule expressed at the surface of erythrocytes infected with mature stages of the M96 strain of P. falciparum that was recognized by a monoclonal antibody (mAb) (TS1/22) directed against an LFA-1 epitope. However, this molecule was not recognized by mAbs directed against other epitopes of LFA-1 or against other integrins. Furthermore, the mAb TS1/22 partially inhibited cytoadherence of parasitized red blood cells to human-brain microvascular endothelial cells. The expression of a molecule sharing an epitope with human LFA-1 integrin on the parasitized erythrocyte surface could be involved in the sequestration of these cells and thus in the pathogenesis of severe disease.     

Evidence for a 6.5-day minimum exoerythrocytic cycle for Plasmodium falciparum in humans and confirmation that immunization with a synthetic peptide representative of a region of the circumsporozoite protein retards infection.

Immunization with a synthetic peptide which is representative of part of the repeating region of Plasmodium falciparum circumsporozoite protein resulted in an immunity which allowed vaccinees to retard the development of patent malaria as compared to nonimmunized controls. Analysis of infection dynamics showed that immunity could be attributed to either neutralization of about 92% of inoculated sporozoites, delayed development of the majority of parasites, or a combination of neutralization and delayed development. In spite of this impressive antiplasmodial capacity, all volunteers after being bitten by infected mosquitoes developed malaria, and seven of eight developed parasitemia between 6.5 and 7.0 days after infective mosquito bites.