Expression of Plasmodium falciparum 3D7 STEVOR proteins for evaluation of antibody responses following malaria infections in naïve infants.

Clinical immunity to Plasmodium falciparum malaria develops after repeated exposure to the parasite. At least 2 P. falciparum variant antigens encoded by multicopy gene families (var and rif) are targets of this adaptive antibody-mediated immunity. A third multigene family of variant antigens comprises the stevor genes. Here, 4 different stevor sequences were selected for cloning and expression in Escherichia coli and His6-tagged fusion proteins were used for assessing the development of immunity. In a cross-sectional analysis of clinically immune adults living in a malaria endemic area in Ghana, high levels of anti-STEVOR IgG antibody titres were determined in ELISA. A cross-sectional study of 90 nine-month-old Ghanaian infants using 1 recombinant STEVOR showed that the antibody responses correlated positively with the number of parasitaemia episodes. In a longitudinal investigation of 17 immunologically naïve 9-month-old infants, 3 different patterns of anti-STEVOR antibody responses could be distinguished (high, transient and low). Children with high anti-STEVOR-antibody levels exhibited an elevated risk for developing parasitaemia episodes. Overall, a protective effect could not be attributed to antibodies against the STEVOR proteins chosen for the study presented here.

The use of anticancer drugs in antiparasitic chemotherapy.

Many similarities exist between cancer cells and parasites. A potentially lucrative starting point for the discovery of novel drugs to combat parasites is to examine available compounds developed against cancer for antiparasitic properties. Here, we review the use of current and promising anticancer agents for treating major human parasitic diseases.

The proteasome inhibitor MLN-273 blocks exoerythrocytic and erythrocytic development of Plasmodium parasites.

Protein degradation is regulated during the cell cycle of all eukaryotic cells and is mediated by the ubiquitin-proteasome pathway. Potent and specific peptide-derived inhibitors of the 20S proteasome have been developed recently as anti-cancer agents, based on their ability to induce apoptosis in rapidly dividing cells. Here, we tested a novel small molecule dipeptidyl boronic acid proteasome inhibitor, named MLN-273 on blood and liver stages of Plasmodium species, both of which undergo active replication, probably requiring extensive proteasome activity. The inhibitor blocked Plasmodium falciparum erythrocytic development at an early ring stage as well as P. berghei exoerythrocytic progression to schizonts. Importantly, neither uninfected erythrocytes nor hepatocytes were affected by the drug. MLN-273 caused an overall reduction in protein degradation in P. falciparum, as demonstrated by immunoblots using anti-ubiquitin antibodies to label ubiquitin-tagged protein conjugates. This led us to conclude that the target of the drug was the parasite proteasome. The fact that proteasome inhibitors are presently used as anti-cancer drugs in humans forms a solid basis for further development and makes them potentially attractive drugs also for malaria chemotherapy.

PCR typing of Plasmodium falciparum in matched peripheral, placental and umbilical cord blood.

A study was carried out in Lambaréné, Gabon, to analyse malarial infections in pregnant women. Blood samples from peripheral circulation and term placentas from 37 women were diagnosed as parasite-positive by thick blood smears. Infection was confirmed by PCR, using single-copy merozoite surface protein 1 and 2 genes. Of the 37 matched cord blood samples, 17 were positive by PCR amplification, even though all but one were microscopically negative. Five of these 17 samples were verified as positive in an antigen detection assay for histidine-rich protein 2. A comparison of alleles from these compartments indicates that single clonal infections were predominant for the cord samples, while double and triple infections were more common for peripheral and placental samples. Knowledge of the occurrence of parasites in each blood sample type is important towards understanding population dynamics in pregnant women and the development of immunity in infants to selected genotypes.

High prevalence of human antibodies to recombinant Duffy binding-like alpha domains of the Plasmodium falciparum-infected erythrocyte membrane protein 1 in semi-immune adults compared to that in nonimmune children.

We used a panel of nine fusion proteins that contain different Duffy binding-like alpha (DBL-alpha) domains of Plasmodium falciparum-infected erythrocyte membrane protein 1 to assess the levels of antibody activity in serum samples obtained from semi-immune or nonimmune individuals from Lambaréné, Gabon. Recognition was measured in terms of either the prevalence or the magnitude of the response. A strong correlation between the immune status of the patients and reactivity with recombinant proteins was observed, which was interpreted as a reflection of the number of infections acquired over time. The antibody responses were predominantly directed toward variable epitopes of the DBL-alpha domain. Antibody responses could be reduced by preincubation of the sera with various fusion proteins. A portion of individuals who exhibited high-level responses to all fusion proteins also had antibodies which recognized conserved epitopes. The possibility that a synergizing effect of anti-DBL-alpha domain antibodies could support chemotherapy is discussed.

Variants of Plasmodium falciparum erythrocyte membrane protein 1 expressed by different placental parasites are closely related and adhere to chondroitin sulfate A.

Plasmodium falciparum-infected erythrocytes adhere to syncytiotrophoblast cells lining the placenta via glycosaminoglycans, such as chondroitin sulfate A (CSA) and hyaluronic acid. Adherence of infected erythrocytes to host receptors is mediated by P. falciparum erythrocyte membrane protein-1 (PfEMP-1). A single PfEMP-1 domain (duffy binding-like [DBL]-3, of the gamma sequence class) from laboratory-adapted strains is thought to be responsible for binding to CSA. In this study, DBL-gamma domains expressed by placental P. falciparum isolates were shown to have an affinity to CSA. All parasite populations accumulating in infected placentas express only 1 variant of PfEMP-1, each of which contains a DBL-gamma domain with CSA binding capacities. Furthermore, sequence analysis data provide evidence for antigenic conservation among the DBL-gamma sequences expressed by different placental parasites. This study offers a close reflection of the process of parasite adhesion in the placenta and is crucial to the understanding of the pathogenesis of malaria during pregnancy.

Molecular cloning of Schistosoma mansoni calcineurin subunits and immunolocalization to the excretory system.

In order to explain the schistosomicidal effect of cyclosporin A, the hypothesis was advanced that the drug, complexed with cyclophilin, inhibits the phosphatase activity of parasite calcineurin (CN), with mechanisms similar to those operating in its immunosuppressive action. As a preparatory step to the testing of this hypothesis, we report the molecular cloning of both CN subunits in Schistosoma mansoni. The catalytic (A) subunit has a predicted sequence of 607 amino acids and shows substantial similarity to other cloned CNs, except for the carboxy-terminal end that is highly divergent. The regulatory (B) subunit consists of 169 amino acids that are 86% identical to those of the human counterpart and, from its anomalous electrophoretic mobility, it appears to be myristoylated. The results of Southern blotting experiments are compatible with the existence of multiple genes for CNA and a single gene for CNB. Western blots showed that both subunits are present at all stages of the parasite life cycle and can be detected both in the soluble and in the membrane fraction. Immunofluorescence confocal microscopy revealed a striking concentration of the anti-CNA reactivity in 6-8 discrete spots in the schistosomula and in distinct spots along the body of the adult parasite, corresponding to the expected localization of flame cells. Both patterns were confirmed by a perfect co-localization of the anti-CNA signal with that of a previously characterized anti-flame cell monoclonal antibody. The preferential confinement of schistosome CN to the protonephridial system suggests that the enzyme in the parasite may fulfil similar functions to those performed in mammalian kidneys.

Schistosoma mansoni: adhesion of mannan-binding lectin to surface glycoproteins of cercariae and adult worms.

Schistosoma mansoni is a blood-dwelling trematode which can persist for several years in the vessels of the human host. The schistosomal surface has been extensively characterized by lectin binding studies, revealing the carbohydrate composition of the worm's tegument. Using fluorescent and scanning electron microscopy we demonstrate that the surface carbohydrates of cercariae and adult worms are the binding ligands for mannanbinding lectin (MBL), a serum protein that is part of the innate immune system. An in vitro complement activation assay with C1q-deficient complement suggests that MBL, in association with the serine proteases MASP-1 and MASP-2, is capable of fixing complement components on the schistosomal tegument and activating the complement cascade via the "MBL pathway." MBL is constitutively expressed by hepatocytes and present in the blood at a stable level. Since it is also a weak acute-phase protein and therefore upregulated in an acute-phase response we investigated the serum MBL levels in patients infected with Schistosoma sp. and in healthy control persons. An enzyme-linked immunosorbent assay indicated no differences between the two groups. Although our results suggest an involvement of MBL activated complement in vitro, its role in vivo remains to be clarified.

[Reinfection with Schistosoma haematobium and mansoni despite repeated praziquantel office treatment in Niger, Mali]. / Réinfections par Schistosoma haematobium et mansoni à l'office du Niger au Mali malgré la prise répétée de praziquantel.

The dynamics of reinfection by Schistosoma haematobium and Schistosoma mansoni after repeated treatment with praziquantel (40 mg/kg body weight, single dose) was studied in a cohort of schoolchildren living in an endemic area. A total of 214 urine and 220 stool samples were collected and examined at three different times, i.e., February 1989, July 1989 and February 1990. Mass chemotherapy was administered at the beginning of study (February 89). Treatment was repeated in children with positive tests at each subsequent sampling. Prevalence rates were 55.1 p. 100, 3.7 p. 100, and 35.0 p. 100 for Schistosoma haematobium and 62.7 p. 100, 46.3 p. 100 and 73.1 p. 100 for Schistosoma mansoni in February 1989, July 1989 and February 1990 respectively (p < 0.001). From July 1989 to February 1990, reinfection was observed in 84.5 p. 100 of children by Schistosoma haematobium versus 57.8 p. 100 by Schistosoma mansoni. The risk of reinfection by Schistosoma haematobium was higher in children between the ages of 7 and 10 years than in children between the ages of 11 and 15 years (p < 0.001), The incidence of intense Schistosoma haematobium egg excretion rose from 0 p. 100 in July 1989 to 6.0 p. 100 in February 1990. The incidence of intense Schistosoma mansoni excretion in February 1990 was 4.5 p. 100. The reinfection rate at 7 months was over 50 p. 100 for both parasite species despite repeated treatment. This finding demonstrates that additional measures such as proper sanitation and vector control are needed to control human schistosomiasis in irrigated rice paddies.

Diagnostic significance of Schistosoma mansoni proteins Sm31 and Sm32 in human schistosomiasis in an endemic area in Egypt.

We performed a series of ELISAs to evaluate the diagnostic significance of two Schistosoma mansoni proteins, Sm31 (cysteine proteinase, cathepsin B) and Sm32 (asparaginyl endopeptidase). Our study populations were chosen from two villages in an endemic area close to Alexandria. Using fusion proteins MS2-Sm31 and MS2-Sm32 as antigens, 70% and 78.9%, respectively, of patient sera from 134 parasitologically confirmed cases reacted positively. The percentage of seropositivity increased to 84.5% when parasite-derived proteins Sm31 and Sm32 were used. The serum levels of antibodies to these two proteins in recombinant or native forms do not correlate with intensity of infection and hence are detected even when egg counts are low, which makes proteins Sm31 and Sm32 useful antigens in the identification of S. mansoni infected cases, particularly in endemic areas in Egypt.

Cyclosporins: lack of correlation between antischistosomal properties and inhibition of cyclophilin isomerase activity.

The immunosuppressive fungal products cyclosporin A (CsA) and FK506 bind with high affinity to intracellular receptor proteins: cyclophilin (CYP) is one of the receptors for CsA and FK506-binding protein (FKBP) is one of the receptors for FK506. These proteins catalyze the in vitro isomerization from a cis to a trans conformation of peptidyl-prolyl bonds in oligopeptides. The relative importance of the peptidyl-prolyl cis-trans isomerase (PPI ase) activity of CYP compared to FKBP in schistosomes is not known. Here, we examine the effects of CsA and FK506 and show that the former inhibits PPIase activity in schistosome extracts, whereas the latter does not. Since CsA is specific for the CYP protein, this result is indicative of the fact that the PPIase activity in the parasite is mostly attributable to CYP. The observation that CsA was significantly more effective than FK506 as an antischistosomal agent, both in vivo and in vitro raises the possibility that killing of schistosomes is caused by the inhibition of schistosome CYP PPIase. We compared a number of Cs analogs for their antischistosomal effects and for the inhibition of CYP PPIase, but were unable to find a correlation between the two properties. We therefore conclude that the lethal effect of CsA is not directly linked to the inhibition of the enzymatic activity of schistosome CYPs.

Expression cloning and biochemical characterizations of recombinant cyclophilin proteins from Schistosoma mansoni.

Recombinant Schistosoma mansoni cyclophilin proteins of the A and the B subtypes (SmCYP A and B) were expressed in bacterial cells as histidine- and maltose-binding fusion proteins and also as nonfused proteins. In addition, S. mansoni CYPs were produced in Sf9 insect cells in their natural forms. Purified recombinant SmCYP B was found to possess a peptidyl-prolyl cis-trans isomerase (PPIase) activity, with a kcat/Km value of 8.2 x 10(5) M-1 s-1. The SmCYP B isoform is approximately two to three times more active than SmCYP A. SmCYP B-specific RNA appears to be more abundant in adult schistosomes than SmCYP A RNA in Northern blots. These results support the conclusion that SmCYP B represents the major schistosomal CYP. The PPIase-associated activity of both CYPs was inhibitable by the immunosuppressive drug cyclosporin A (CsA). We attempt to explain differences in PPIase activities and in CsA inhibition by examining models of the two CYPs complexed to CsA.

Recognition of Schistosoma mansoni cathepsins B and L by human IgG1 and IgG4 antibodies.

Human schistosome infections are chronic in nature and elevated IgG4 levels are associated with length of exposure. To examine how structure, localization and dynamics of exposure of a protein to the immune system can affect antibody isotype expression, specific antibody levels against two Schistosoma mansoni recombinant cathepsin molecules were determined in S. mansoni-infected individuals. Cathepsin B (rCatB, secreted in the gut) and cathepsin L (rCatL, localized in the reproductive structures) were used to determine IgG1 reactivity, as a measure of the stimulation of the immune response, and the switch to IgG4 as an indicator of the dynamics and rate of presentation to the immune system. Sera from three groups of S. mansoni-infected patients were used: individuals with life-long exposure in an endemic area in Burundi, a group from a recent endemic focus in Senegal, and of acutely infected European travellers. We report for the first time the ability of rCatL to trigger an immune response and show distinct antibody isotype reactivity between rCatB versus rCatL. Patients harbouring long-term chronic infections had elevated levels of IgG4 to both antigen, whereas individuals infected for less than four years had high IgG4 to rCatB but not to rCatL. Acutely infected travellers developed IgG1 to rCatB only. Our results demonstrate that an immune response is mounted more rapidly against a cathepsin molecule secreted in the gut of the worm than against an internally localized cathepsin.

Biochemical properties of purified cathepsin B from Schistosoma mansoni.

A previously described "major acidic proteinase" of adult Schistosoma mansoni, believed to play a key role in the parasite's metabolism, has been identified as a cathepsin B (Sm31). Purified Sm cathepsin B was not recognized by anti-Sm32 or anti-cathepsin L antibodies. The enzyme hydrolyzes the synthetic protease substrates Z-Arg-Arg-AMC and Z-Phe-Arg-AMC as well as protein substrates. Its pH optimum is 3.0 with serum albumin, 4.0-5.0 with globin and 5.5-6.0 with the synthetic substrates. The enzyme was inactivated by cysteine proteinase inhibitors. Its activity against protein substrates would support the hypothesis that it plays a role in schistosome nutrition.

Characterization of a Schistosoma mansoni cDNA encoding a B-like cyclophilin and its expression in Escherichia coli.

A cDNA encoding a Schistosoma mansoni cyclophilin (SmCyP) has been cloned by polymerase chain reaction amplification using degenerate oligonucleotides based on known conserved cyclophilin (CyP) sequences and by screening an expression cDNA library. The cDNA sequence encodes a 21.5-kDa protein, which shares 59% sequence identity with human CyP B. The SmCyP protein was expressed in Escherichia coli with a hexahistidine affinity tag at its amino terminus and antibodies to the purified (His6)-SmCyP fusion protein were raised in a rabbit. Fractionation of parasite material followed by immunoblot analysis revealed that schistosome CyP is a soluble protein. The N-terminus of the predicted protein contains a hydrophobic region, suggestive of a signal sequence. Accordingly, a recombinant SmCyP protein, lacking the first 23 amino acids was found to share the same gel electrophoretic mobility as the parasite-derived CyP protein, suggesting cleavage of a leader sequence. Hybridization of genomic DNA to a full-length cDNA probe indicates that the SmCyP gene is present as a single copy. Immunohistological experiments in conjunction with confocal scanning laser microscopy and immune electron microscopy show that SmCyP is present in abundance in the adult worm as well as in the schistosomula. The function of CyP in the schistosome is presently unclear, but since its ligand, cyclosporin A, has antischistosomal activity, its function is expected to be a vital one.

Sequence, characterization and localization of a cysteine proteinase cathepsin L in Schistosoma mansoni.

A cDNA encoding Schistosoma mansoni cathepsin L was isolated from a cDNA library and sequenced. Alignment of the proposed amino-acid sequence with known members of cathepsin L shows highest homologies with sequences from mouse and rat. An expression plasmid was constructed in Escherichia coli to produce recombinant schistosome cathepsin L with an extension of six histidines at its N terminus. Using antibodies raised against the purified fusion protein, two polypeptide bands with approx. molecular masses of 38 and 31 kDa were identified in a schistosome extract. By use of specific radioiodinated inhibitors, a radioactively labeled protein could be detected at 31 kDa, suggesting that this is the active mature enzyme. The larger protein of 38 kDa did not react with the inhibitor, indicating that it represents the inactive precursor molecule. Immunohistological experiments revealed that the proteinase is localized in structures associated with the reproductive system of females and with the subtegumental region of the gynecophoric canal of males. However, Northern blot hybridization demonstrates that more transcripts are present in female parasites than in males. Genomic Southern blotting suggests that schistosome cathepsin L is expressed from a single-copy gene.

Sequence and structure similarities of cathepsin B from the parasite Schistosoma mansoni and human liver.

A three-dimensional structure of Schistosoma mansoni cathepsin B was modelled using the coordinates of the crystal structure of the human liver enzyme. Both enzymes appear to share remarkable structural similarity. However, an examination of the models complexed with two synthetic inhibitors revealed differences in inhibitor binding, as confirmed by differences in the 50% inhibitory concentration of the same inhibitor.

Expression and partial characterization of a cathepsin B-like enzyme (Sm31) and a proposed 'haemoglobinase' (Sm32) from Schistosoma mansoni.

Schistosoma mansoni protein Sm31 is a cysteine proteinase similar to mammalian lysosomal cathepsin B, proposed to be a key enzyme in schistosome metabolism. Protein Sm32 has been identified as a putative cysteine proteinase termed a 'haemoglobinase'. Since neither Sm31 nor Sm32 have been completely purified, some controversy of the nature of the 'true' digestive enzyme still exists. By incubating a radiolabelled cysteine-proteinase active-site-directed synthetic inhibitor with total S. mansoni proteins, the target of inhibition was Sm31 and not Sm32. The selectivity and irreversibility of inactivation make affinity labelling an invaluable tool for exploring key differences among closely related enzymes and also for studying proteinase activity in a cellular environment. In order to confirm these results, we expressed the complete cDNA sequences of Sm31 and Sm32 in insect cells and analysed the recombinant gene products for proteolytic activities. Cell extracts containing S. mansoni cathepsin B, but not those expressing 'haemoglobinase', were demonstrated to cleave a synthetic substrate benzyloxycarbonyl-arginylarginylaminomethylcoumarin in fluorescence assays. Our findings confirm previous assertions that a cysteine proteinase resembling cathepsin B is the haemoglobinase involved in digestion of host proteins. Thus, the original proposal that Sm32 is a cysteine proteinase has not been verified, and its function remains unknown.