Yvon Sterkers

Immunization with Recombinant Duffy Binding-Like-γ3 Induces Pan-Reactive and Adhesion-Blocking Antibodies against Placental Chondroitin Sulfate A-Binding Plasmodium falciparum Parasites

Maternal malaria is associated with the sequestration, in the placenta, of Plasmodium falciparum-infected erythrocytes onto chondroitin sulfate A (CSA), via the duffy binding-like (DBL)-gamma3 domain of the P. falciparum erythrocyte membrane protein 1 (PfEMP1(CSA)) (DBL-gamma3(CSA)). The production of antibodies against CSA-binding infected erythrocytes (IEs(CSA)) is correlated with resistance to maternal malaria in multiparous women. We produced recombinant DBL-gamma3(CSA) (rDBL-gamma3(CSA)) in insect cells, corresponding to 2 variant DBL-gamma3(CSA) subtypes that mediate binding to CSA in laboratory lines and placental isolates. Both recombinant cysteine-rich DBL-gamma3(CSA) domains blocked IEs(CSA) binding to CSA. Immunization of mice, with the rDBL-gamma3(CSA)-FCR3 and rDBL-gamma3(CSA)-3D7 domains, resulted in the generation of antibodies recognizing homologous and heterologous rDBL-gamma3(CSA), a finding indicating conserved epitopes inducing a pan-reactive immune response. Mouse monoclonal antibodies (MAbs) against both recombinant proteins were pan-reactive with various IEs(CSA). One MAb efficiently inhibited and reversed IE(CSA) cytoadhesion to endothelial cells in vitro. Thus, DBL-gamma3(CSA) is the target of inhibitory and pan-reactive antibodies. Saimiri sciureus monkeys immunized with FCR3-rDBL-gamma3(CSA) developed pan-reactive and inhibitory antibodies, a finding suggesting that the development of a vaccine to prevent maternal malaria is feasible.

Constitutive mosaic aneuploidy is a unique genetic feature widespread in the Leishmania genus.

Using fluorescence in situ hybridization, we determined the ploidy of four species of Leishmania: Leishmania infantum, Leishmania donovani, Leishmania tropica and Leishmania amazonensis. We found that each cell in a strain possesses a combination of mono-, di- and trisomies for all chromosomes; ploidy patterns were different among all strains/species. These results extend those we previously described in Leishmania major, demonstrating that mosaic aneuploidy is a genetic feature widespread to the Leishmania genus. In addition to the genetic consequences induced by this mosaicism, the apparent absence of alternation between haploid/diploid stages questions the modality of genetic exchange in Leishmania sp.

Investigation of host factors possibly enhancing the emergence of the chondroitin sulfate A-binding phenotype in Plasmodium falciparum

In malaria endemic areas, regardless of immunity acquired during lifelong exposure to malaria, pregnant women become susceptible to Plasmodium falciparum infections. Malaria during pregnancy is associated with a massive sequestration of infected erythrocytes in the placenta and the emergence of a unique parasite-derived adhesive molecule (encoded by var2CSA) that binds to chondroitin sulfate A (CSA). How P. falciparum achieves the timely expression of the CSA ligand in pregnant women remains puzzling. We investigated whether host serum-specific factors present only during pregnancy may induce var2CSA expression. Our panel of experiments did not reveal significant changes in var2CSA levels and CSA-binding capacity.

TbFlabarin, a flagellar protein of Trypanosoma brucei, highlights differences between Leishmania and Trypanosoma flagellar-targeting signals

TbFlabarin is the Trypanosoma brucei orthologue of the Leishmania flagellar protein LdFlabarin but its sequence is 33% shorter than LdFlabarin, as it lacks a C-terminal domain that is indispensable for LdFlabarin to localize to the Leishmania flagellum. TbFlabarin is mainly expressed in the procyclic forms of the parasite and localized to the flagellum, but only when two palmitoylable cysteines at positions 3 and 4 are present. TbFlabarin is more strongly attached to the membrane fraction than its Leishmania counterpart, as it resists complete solubilization with as much as 0.5% NP-40. Expression ablation by RNA interference did not change parasite growth in culture, its morphology or apparent motility. Heterologous expression showed that neither TbFlabarin in L. amazonensis nor LdFlabarin in T. brucei localized to the flagellum, revealing non-cross-reacting targeting signals between the two species.

Evaluation of five automated and one manual methods for Toxoplasma and human DNA extraction from artificially spiked amniotic fluid

OBJECTIVESnMolecular detection of Toxoplasma gondii plays a crucial role in the prenatal and neonatal diagnosis of congenital toxoplasmosis (CT). Sensitivity of this diagnosis is partly related to the efficiency of parasite DNA extraction and amplification. DNA extraction methods with automated platforms have been developed. Therefore, it is essential to evaluate them in combination with adequate PCR amplification assays.nnnMETHODSnIn this multisite study, we investigated the suitability of two recent automated procedures for the isolation of Toxoplasma DNA from amniotic fluid (AF) (Magtration system 12GC, PSS and Freedom EVO VacS, Tecan), compared with three other automated procedures (MagNAPure Compact, Roche, BioRobot EZ1, Qiagen and modified NucliSens easyMAG, bioMérieux) and with the manual DNA extraction QIAamp DNA Mini kit (Qiagen). Two Toxoplasma PCR assays targeting the 529-bp repeat DNA element were used, based upon dual hybridization (FRET) or hydrolysis (TaqMan) probes. A total of 1296 PCRs were performed including 972 Toxoplasma PCRs.nnnRESULTSnWe showed variable efficacy (4.2%-100% positive results) among the DNA extraction procedures in isolating up to five T.xa0gondii cells/mL in AF samples. Moreover, for a given DNA extraction method, variable results were obtained among the two Toxoplasma PCR assays for detecting up to five T.xa0gondii cells/mL: when using TaqMan PCR, all the automated systems yielded more than 60% positive results. Nevertheless, when testing the DNA extracts in triplicate, four out of six extraction methods allowed a satisfactory detection of low amounts of T.xa0gondii DNA (≥33% of positive results) independently of the PCR assay used.nnnCONCLUSIONSnDespite the influence of the subsequent PCR method used, this study should help microbiologists in the choice of DNA extraction methods for the detection of T.xa0gondii in amniotic fluid. The extraction method should be checked as adequate for the PCR assay used.

Quality Control for the Molecular Diagnosis of Toxoplasmosis

Toxoplasmosis is an endemic parasitic disease due to the protozoon Toxoplasma gondii. The definitive host is the cat, in which the parasite develops in the intestinal epithelium, before being eliminated as oocysts in the faeces. When oocysts, for example in contaminated soil, are ingested by humans, other mammals or birds, they pass through the stomach and excyst in the intestine; the released sporozoites invade the intestinal wall and give rise to generalized infection (Remington & Desmonts, 1995; Wong & Remington, 1993). Two tissue stages are important in pathogenesis: tachyzoites and bradyzoites. Initially, the infection consists mainly of rapidly dividing tachyzoites. Then, with developing immunity, persistent tissue cysts containing hundreds of slowly multiplying bradyzoites develop, especially in the muscle and brain. This ability to live inside cells without destroying them allows evasion of host immunity and the development of a chronic stage of infection, lasting for years. Intermediate hosts become infected either by ingesting soil, water or foodstuff contaminated with cat faeces or by ingesting raw or undercooked meat containing tissue cysts. T. gondii is responsible for generally benign infections except when the disease occurs in pregnant women (congenital toxoplasmosis) or in immunocompromised individuals, such as human immunodeficiency virus-positive or grafted patients, in which cases the vital prognosis may be involved. In certain countries of Europe, including France, toxoplasmosis is regarded as a serious health problem. In France, the prevalence of acquired toxoplasmosis in adults is 44%, and the estimated yearly incidence of contamination in women during pregnancy and of congenital toxoplasmosis are high, respectively, 6-7/1000 and 0.1% of births (Berger et al., 2008, King et al., 2008). Prevention of congenital toxoplasmosis (CT), including prenatal diagnosis (PND), has become a national policy in France since 1978 (Thulliez, 1992). Serological screening and follow-up is established for non-immunized pregnant women, and associated with monthly ultrasound examinations. PND is proposed in case of seroconversion between 6 to 38 weeks of amenorrhea. For this, PCR-based molecular diagnostic tests using amniotic fluid, introduced in the early 90s, have rapidly eliminated the need for cordocentesis, and have competed with more classical biological methods. Wherever it has been implemented, PND of CT has clearly improved the prognosis and outcome of infected children (reviewed in (Bastien, 2002)).