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We studied all consensus sequences within the four least 'variable blocks' (VB) present in the DBL6 epsilon domain of VAR2CSA, the protein involved in the adhesion of infected red blood cells by Plasmodium falciparum that causes the Pregnancy-Associated Malaria (PAM). Characterising consensus sequences with respect to recognition of antibodies and percentage of responders among pregnant women living in areas where P. falciparum is endemic allows the identification of the most antigenic sequences within each VB. When combining these consensus sequences among four serotypes from VB1 or VB5, the most often recognized ones are expected to induce pan-reactive antibodies recognizing VAR2CSA from all plasmodial strains. These sequences are of main interest in the design of an immunogenic molecule. Using a similar approach than for DBL6e, we studied the five other DBL and the CIDRpam from VAR2CSA, and again identified VB segments with highly conserved consensus sequences. In addition, we identified consensus sequences in other var genes expressed by non-PAM parasites. This finding paves the way for vaccine design against other pathologies caused by P. falciparum.

Background: Haptoglobin related protein (Hpr) is a key component of trypanosome lytic factors (TLF), a subset of high-density lipoproteins (HDL) that form the first line of human defence against African trypanosomes. Hpr, like haptoglobin (Hp) can bind to hemoglobin (Hb) and it is the Hpr-Hb complexes which bind to these parasites allowing uptake of TLF. This unique form of innate immunity is primate-specific. To date, there have been no population studies of plasma levels of Hpr, particularly in relation to hemolysis and a high prevalence of ahaptoglobinemia as found in malaria endemic areas. Methods and Principal Findings: We developed a specific enzyme-linked immunosorbent assay to measure levels of plasma Hpr in Gabonese children sampled during a period of seasonal malaria transmission when acute phase responses (APR), malaria infection and associated hemolysis were prevalent. Median Hpr concentration was 0.28 mg/ml (range 0.03-1.1). This was 5-fold higher than that found in Caucasian children (0.049 mg/ml, range 0.002-0.26) with no evidence of an APR. A general linear model was used to investigate associations between Hpr levels, host polymorphisms, parasitological factors and the acute phase proteins, Hp, C-reactive protein (CRP) and albumin. Levels of Hpr were associated with Hp genotype, decreased with age and were higher in females. Hpr concentration was strongly correlated with that of Hp, but not CRP. Conclusions/Significance: Individual variation in Hpr levels was related to Hp level, Hp genotype, demographics, malaria status and the APR. The strong correlations between plasma levels of Hp and Hpr suggest that they are regulated by similar mechanisms. These population-based observations indicate that a more dynamic view of the relative roles of Hpr and Hpr-Hb complexes needs to be considered in understanding innate immunity to African trypanosomes and possibly other pathogens including the newly discovered Plasmodium spp of humans and primates.

Purpose of the research: VAR2CSA is considered as the main target of protective immunity against pregnancy-associated malaria. VAR2CSA high molecular weight complicates scaling up production of VAR2CSA recombinant protein for large-scale vaccination programmes. We previously demonstrated that antibodies induced by NTS-DBL1X-Id1-DBL2X efficiently block parasite binding to CSA in a similar manner to antibodies induced by the full-length extracellular part of VAR2CSA. In order to identifying the shortest fragment of VAR2CSA carrying major protective epitopes able to elicit inhibitory antibodies, we performed a refined antigenic mapping of NTS-DBL1X-Id1-DBL2X through a DNA vaccination technique. Principal results: Five single or double domains constructs encoding NTS-DBL1X, NTS-DBL1X-Id1, Id1, Id1-DBL2X and DBL2X were made and used to immunize mice. The NTS-DBL1X, NTS-DBL1X-Id1, and Id1-DBL2X fragments all raised high titer immune response, as measured by ELISA. The DBL2X fragment raised a weaker antibody titer, and the Id1 construct failed to elicit antibody. Sera from mice immunized with NTS-DBL1X or DBL2X constructs failed to block infected erythrocytes binding to CSA, whereas sera from mice immunized with NTS-DBL1X-Id1 showed partial inhibitory activity, and the Id1-DBL2X fragment elicited antisera that totally abrogated infected erythrocytes adhesion to CSA. IgG purified from Id1-DBL2X antisera showed a similar inhibitory profile than Id1-DBL2X antisera. Anti-FCR3 anti-Id1-DBL2X antibodies also efficiently block the adhesion of erythrocytes infected by the HB3 parasite line to CSA. Id1-DBL2X antisera recognized the surface of field isolates from pregnant women, and inhibited CSA-binding of all 8 isolates tested, although to a variable level. Major conclusions: We raised high-titer antibodies against several parts of the protein, and identified Id1-DBL2X as the minimal VAR2CSA fragment inducing antibodies with CSA-binding inhibitory efficiency in the same range as the full-length extracellular part of VAR2CSA.

High antibody levels directed to Plasmodium falciparum merozoite surface proteins (MSP), including MSP2, as well as genetically related red blood cell defects, have previously been found to be associated with protection against malaria. Here, our main objective was to study the changes in MSP2-specific total IgG, IgG1 and IgG3 responses during a malaria transmission season in order to assess the impact of sickle-cell, alpha(+)-thalassemia and G6PD variants on antibody kinetics. Repeated parasitological assessments of a cohort of children were conducted during an 8-month period. Antibody responses to recombinant MSP2/3D7 and MSP2/FC27 proteins were measured at the beginning and at the end of transmission season. We found that (i) the period of last Plasmodium falciparum infection during the transmission season was associated with IgG3 anti-MSP2 change. Compared to the IgG3 levels of children infected in January 2003 (end of transmission season), the IgG3 level of children decreased with the length of the period without infection, (ii) G6PD A carriers had a lower increase of IgG3 levels to MSP2/FC27 and MSP2/3D7 during the transmission season than the noncarriers. This latter finding is suggestive of qualitative and/or quantitative reduction of exposure to malarial antigens related to this genetic variant, leading to weaker stimulation of specific antibody responses. We speculate that cellmediated immune activity may explain the clinical protection afforded by this genetic trait.

Pregnancy-associated malaria (PAM) arises from sequestration of Plasmodium falciparum-parasitized erythrocytes (PE) in the placenta, leading to chronic symptoms in the expectant mother and serious consequences for fetal development. Placental sequestration has been linked to binding of chondroitin sulphate A (CSA) by the var2CSA variant of PfEMP1 expressed on the PE surface, and a substantial body of evidence shows that the immune response to var2CSA gives an effective protection against PAM. We have expressed the var2CSA-DBL5 epsilon domain, derived from a placental isolate from Senegal, as soluble product in Escherichia coil and have shown using different criteria that the recombinant protein is obtained with the native conformation. Using surface plasmon resonance techniques, we have examined binding of DBL5 epsilon to placental chondroitin sulphate proteoglyan and CSA; however, the recombinant protein also binds to other sulphated oligosaccharides, with higher affinity in some cases, indicating that the single domain lacks the specificity for CSA shown by the complete extra-cellular region of var2CSA and placental parasites. Recombinant DBL5 epsilon was specifically recognized by sera from malaria-exposed Senegalese women in a parity-dependent manner but by sera not from children or males from the same endemic region. Conversely, DBL5 epsilon induced antibodies in mice that recognized placental isolates from Benin but not isolates from children. The presence of universal epitopes thus supports DBL5 epsilon as an interesting component of var2CSA to be considered for vaccine development.

Background. We compared sulfadoxine-pyrimethamine (SP) with unsupervised artemether-lumefantrine (AL) and unsupervised amodiaquine-artesunate (ASAQ) fixed-dose formulation for the treatment of uncomplicated malaria in children in Benin. Methods. This open-label, noninferiority comparative trial included children aged 6-60 months. The follow-up period was 6 weeks, and the primary objective was a comparison of polymerase chain reaction (PCR)-adjusted effectiveness rates at day 28. Results. The study included 240 children (48 received SP, and 96 each received AL and ASAQ). The intention-to-treat analysis showed effectiveness rates on day 28 of 20.8%, 78.1%, and 70.5% for SP, AL, and ASAQ, respectively. After adjustment for PCR results, these rates were 27.1%, 83.3%, and 87.4%, respectively. The per-protocol analysis (217 patients) showed effectiveness rates on day 28 of 21.7%, 88.0%, and 76.1% for SP, AL, and ASAQ, respectively. After adjustment for PCR results, these rates were 28.3%, 94.0%, and 93.2%, respectively. SP was less effective than the other drugs in the PCR-adjusted analysis, whereas AL and ASAQ were equally effective. The rate of new infection was higher among children treated with ASAQ than among those treated with AL. Conclusions. This was the first trial, to our knowledge, to compare unsupervised AL with unsupervised ASAQ fixed-dose formulation; both treatments provided high PCR-adjusted day 28 effectiveness rates. Efficacy rates for SP were surprisingly low.

Background: Pregnant women develop protective anti-VSA IgG1 and IgG3 when infected by Plasmodium falciparum. The major target of IgG from serum of infected pregnant women is VAR2CSA. Methods: In this study, ELISA was used to compare the level of VAR2CSA DBL5 epsilon- specific IgG subclasses at enrolment and at delivery in a cohort of pregnant women in Senegal. All antibody measures were analysed in relation to placental infection according to parity. Results: The results show an interaction between immune response to placental malaria and parity. A higher level of anti-DBL5 epsilon-IgG3 at enrolment and a higher increase between enrolment and delivery were found in primigravidae who presented with uninfected placenta at delivery in comparison to those who presented with an infection of the placenta. However, high antibody level at delivery was associated with the infection of the placenta in multigravidae. Conclusion: This high level of IgG3 in uninfected primigravidae suggests a protective role of these antibodies in this susceptible group, highlighting the importance of VAR2CSA in general and of some of its variants still to be defined, in the induction of protective immunity to pregnancy malaria.

Different domains of a novel full-length var gene (termed 732var) isolated from a placenta of a malaria-infected woman were expressed in Escherichia coli as recombinant proteins and analysed biochemically and immunologically. Two of these, the Duffy binding-like (DBL)-3 gamma domain and the cysteine-rich interdomain region (CIDR)-1 alpha were able to bind chondroitin sulfate A and CD36, respectively. The DBL-3 gamma domain was investigated in a previous study and confirmed here to exhibit anti-disease characteristics related to pregnancy-associated malaria. Mothers with high anti-DBL-3 gamma antibody levels were protected from placental infection. The novel finding in this study is that babies born to mothers carrying anti-CIDR-1 alpha antibodies had a delayed time to the first infection.

Placental Plasmodium falciparum sequestration is associated with dysregulated immune function. Placental inflammatory responses via IFN-gamma and TNF-alpha are implicated in functional damage. However, they are needed during placental infection to control asexual stage parasites. To test the hypothesis that placental immunomodulation associated with malaria disturbs cytokine secretion differently in monocytes and lymphocytes, we have determined the proportion of monocytes and/or lymphocytes secreting IFN-gamma, TNF-alpha, IL-10 and IL-12. Intervillous and peripheral blood monocyte (CD14(+)) and lymphocyte (CD3/CD4(+); CD3/CD8(+)) cytokine production was compared between 17 P. falciparum-infected and 12 non-infected Senegalese women. After culture with phorbolmyristate acetate/ionomycin (PMA/iono), lipopolysaccharide (LPS) or P. falciparum-infected erythrocytes (IE), the intracellular expression of cytokines in lymphocytes (IFN-gamma, TNF-alpha) and monocytes (IL-10, IL-12, TNF-alpha), was detected. In response to IE, CD4(+) and CD8(+) T-cells produced IFN-gamma and TNF-alpha at similar rates in both compartments. In response to PMA/iono, the frequencies of CD4(+) and CD8(+) T-cells producing IFN-gamma and TNF-alpha were similar in both compartments, but increased in P. falciparum-infected placentas. In response to LPS or IE, IL-12 secreting monocytes were increased in infected women, while the frequency of TNF-alpha secreting monocytes was decreased compared to that in non-infected placenta. The monocyte IL-12 response is not impaired in infected women. IL- 12 is an important factor for inducing IFN-gamma in T-cells. Thus, 1L-12 and IFN-alpha responses may synergistically allow a protective immune response in placental malaria. TNF-alpha production by CD4(+) and CD8(+) T-cells, is up-regulated in P falciparum-infected placentas, suggesting that T-cells actively participate to inflammatory responses.

We have previously identified a number of DBL gamma domains in Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) transcripts obtained from placental parasite isolates, showing that they bind specifically to chondroitin sulfate A (CSA) (Khattab A, Kun J, Deloron P, Kremsner PG, Klinkert MQ. Variants of Plasmodium falciparum erythrocyte membrane protein I expressed by different placental parasites are closely related and adhere to chondroitin sulfate A. J Infect Dis 2001;183:1165-9). Here we give a more detailed physico-chemical and binding characterisation of the soluble, recombinant DBL gamma domain derived from one of these isolates. Results from circular dichroism and limited proteolysis experiments are consistent with the recombinant domain being expressed with the native fold. Specific binding of DBL gamma to placental cryosections was demonstrated by labeling with antibodies raised against the recombinant domain; binding was diminished after treatment of the cryosections with chondroitinase or by blocking with anti-CSA antibody, showing that CSA mediates the interaction. Binding of the DBL gamma domain to purified placental chondroitin sulfate proteoglycan (CSPG) was also studied using surface plasmon resonance techniques, with DBL gamma as analyte and CSPG immobilised on the sensor chip; these quantitative measurements gave an affinity constant in the mu-molar range under the conditions used. The native conformation of the DBL gamma domain is essential for CSPG recognition since binding to the sensor chip is abolished when the protein is irreversibly reduced. As with the placental cryosections, association was significantly reduced after treating the immobilised CSPG with chondroitinase. Together, these results demonstrate specific interaction between the DBL gamma domain and the placental receptor.