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Background Plasmodium falciparum infections are especially severe in pregnant women because infected erythrocytes (IE) express VAR2CSA, a ligand that binds to placental trophoblasts, causing IE to accumulate in the placenta. Resulting inflammation and pathology increases a woman’s risk of anemia, miscarriage, premature deliveries, and having low birthweight (LBW) babies. Antibodies (Ab) to VAR2CSA reduce placental parasitaemia and improve pregnancy outcomes. Currently, no single assay is able to predict if a woman has adequate immunity to prevent placental malaria (PM). This study measured Ab levels to 28 malarial antigens and used the data to develop statistical models for predicting if a woman has sufficient immunity to prevent PM. Methods Archival plasma samples from 1377 women were screened in a bead-based multiplex assay for Ab to 17 VAR2CSA-associated antigens (full length VAR2CSA (FV2), DBL 1-6 of the FCR3, 3D7 and 7G8 lines, ID1-ID2a (FCR3 and 3D7) and 11 antigens that have been reported to be associated with immunity to P. falciparum (AMA-1, CSP, EBA-175, LSA1, MSP1, MSP2, MSP3, MSP11, Pf41, Pf70 and RESA)). Ab levels along with clinical variables (age, gravidity) were used in the following seven statistical approaches: logistic regression full model, logistic regression reduced model, recursive partitioning, random forests, linear discriminant analysis, quadratic discriminant analysis, and support vector machine. Results The best and simplest model proved to be the logistic regression reduced model. AMA-1, MSP2, EBA-175, Pf41, and MSP11 were found to be the top five most important predictors for the PM status based on overall prediction performance. Conclusions Not surprising, significant differences were observed between PM positive (PM+) and PM negative (PM−) groups for Ab levels to the majority of malaria antigens. Individually though, these malarial antigens did not achieve reasonably high performances in terms of predicting the PM status. Utilizing multiple antigens in predictive models considerably improved discrimination power compared to individual assays. Among seven different classifiers considered, the reduced logistic regression model produces the best overall predictive performance. Electronic supplementary material The online version of this article (doi:10.1186/s12936-017-2041-3) contains supplementary material, which is available to authorized users.

Background Antigenic variation of Plasmodium falciparum erythrocyte membrane protein 1 is a key parasite mechanism for immune evasion and parasite survival. It is assumed that the number of parasites expressing the same var gene must reach high enough numbers before the host can produce detectable levels of antibodies (Ab) to the variant. VAR2CSA is a protein coded for by one of 60 var genes that is expressed on the surface of infected erythrocytes (IE) and mediates IE binding to the placenta. The idea that Ab to VAR2CSA are pregnancy-associated was challenged when VAR2CSA-specific Ab were reported in children and men. However, the frequency and conditions under which Ab to VAR2CSA are produced outside pregnancy is unclear. This study sought to determine frequency, specificity and level of Ab to VAR2CSA produced in children and whether children with hyperparasitaemia and severe malaria are more likely to produce Ab to VAR2CSA compared to healthy children. Methods Antibody responses to a panel of recombinant proteins consisting of multiple VAR2CSA Duffy-binding-like domains (DBL) and full-length VAR2CSA (FV2) were characterized in 193 1–15 year old children from rural Cameroonian villages and 160 children with severe malaria from the city. Results Low Ab levels to VAR2CSA were detected in children; however, Ab levels to FV2 in teenagers were rare. Children preferentially recognized DBL2 (56–70%) and DBL4 (50–60%), while multigravidae produced high levels of IgG to DBL3, DBL5 and FV2. Sixty-seven percent of teenage girls (n = 16/24) recognized ID1–ID2a region of VAR2CSA. Children with severe forms of malaria had significantly higher IgG to merozoite antigens (all p 0.05) when compared to the healthy children. Conclusion The study suggests that children, including teenage girls acquire Ab to VAR2CSA domains and FV2, but Ab levels are much lower than those needed to protect women from placental infections and repertoire of Ab responses to DBL domains is different from those in pregnant women. Interestingly, children with severe malaria did not have higher Ab levels to VAR2CSA compared to healthy children. Electronic supplementary material The online version of this article (doi:10.1186/s12936-016-1585-y) contains supplementary material, which is available to authorized users.

Background Placental malaria occurs when Plasmodium falciparum infected erythrocytes sequester in the placenta. Placental parasite isolates bind to chondroitin sulphate A (CSA) by expression of VAR2CSA on the surface of infected erythrocytes, but may sequester by other VAR2CSA mediated mechanisms, such as binding to immunoglobulins. Furthermore, other parasite antigens have been associated with placental malaria. These findings have important implications for placental malaria vaccine design. The objective of this study was to adapt and describe a biologically relevant model of parasite adhesion in intact placental tissue. Results The ex vivo placental perfusion model was modified to study adhesion of infected erythrocytes binding to CSA, endothelial protein C receptor (EPCR) or a transgenic parasite where P. falciparum erythrocyte membrane protein 1 expression had been shut down. Infected erythrocytes expressing VAR2CSA accumulated in perfused placental tissue whereas the EPCR binding and the transgenic parasite did not. Soluble CSA and antibodies specific against VAR2CSA inhibited binding of infected erythrocytes. Conclusion The ex vivo model provides a novel way of studying receptor-ligand interactions and antibody mediated inhibition of binding in placental malaria. Electronic supplementary material The online version of this article (doi:10.1186/s12936-016-1342-2) contains supplementary material, which is available to authorized users.

Background In pregnant women, P. falciparum-infected erythrocytes (IEs) express a unique member of the PfEMP1 family named VAR2CSA, which is associated with the ability of the IEs to adhere to chondroitin sulphate A (CSA) in the placenta [1]. Understanding the mechanism behind this specific CSA interaction is important for the optimal design of a vaccine against placental malaria. CSAbinding of single domains of VAR2CSA has appeared to not reflect the specificity of the native IE binding [2].

Background Malaria caused by Plasmodium falciparum can result in several different syndromes with severe clinical consequences for the about 200 million individuals infected each year. During pregnancy, women living in endemic areas become susceptible to malaria due to lack of antibodies against a unique P. falciparum membrane protein, named VAR2CSA. This antigen is not expressed in childhood infections, since it binds chondroitin sulphate A (CSA) expressed on the intervillous space in the placenta. A vaccine appears possible because women acquire protective antibodies hindering sequestration in the placenta as a function of parity. A challenge for vaccine development is to design small constructs of this large antigen, which can induce broadly protective antibodies. It has previously been shown that one domain of VAR2CSA, DBL4-FCR3, induces parasite adhesion-blocking antibodies. In this study, it is demonstrated that other domains of VAR2CSA also can induce antibodies with inhibitory activity. Methods All VAR2CSA domains from the 3D7 and HB3 parasites were produced in Baculovirus-transfected insect cells. Groups of three rats per protein were immunized and anti-sera were tested for surface reactivity against infected erythrocytes expressing FCR3 VAR2CSA and for the ability to inhibit FCR3CSA parasite adhesion to CSA. The fine specificity of the immune sera was analysed by VAR2CSA peptide arrays. Results Inhibitory antibodies were induced by immunization with DBL3-HB3 T1 and DBL1-3D7. However, unlike the previously characterised DBL4-FCR3 response the inhibitory response against DBL1-3D7 and DBL3-HB3 T1 was poorly reproduced in the second rounds of immunizations. Conclusion It is possible to induce parasite adhesion-blocking antibodies when immunizing with a number of different VAR2CSA domains. This indicates that the CSA binding site in VAR2CSA is comprised of epitopes from different domains.

Background Pregnancy malaria is caused by Plasmodium falciparum-infected erythrocytes binding the placental receptor chondroitin sulfate A (CSA). This results in accumulation of parasites in the placenta with severe clinical consequences for the mother and her unborn child. Women become resistant to placental malaria as antibodies are acquired which specifically target the surface of infected erythrocytes binding in the placenta. VAR2CSA is most likely the parasite-encoded protein which mediates binding to the placental receptor CSA. Several domains have been shown to bind CSA in vitro; and it is apparent that a VAR2CSA-based vaccine cannot accommodate all the CSA binding domains and serovariants. It is thus of high priority to define minimal ligand binding regions throughout the VAR2CSA molecule. Methods To define minimal CSA-binding regions/peptides of VAR2CSA, a phage display library based on the entire var2csa coding region was constructed. This library was screened on immobilized CSA and cells expressing CSA resulting in a limited number of CSA-binding phages. Antibodies against these peptides were affinity purified and tested for reactivity against CSA-binding infected erythrocytes. Results The most frequently identified phages expressed peptides residing in the parts of VAR2CSA previously defined as CSA binding. In addition, most of the binding regions mapped to surface-exposed parts of VAR2CSA. The binding of a DBL2X peptide to CSA was confirmed with a synthetic peptide. Antibodies against a CSA-binding DBL2X peptide reacted with the surface of infected erythrocytes indicating that this epitope is accessible for antibodies on native VAR2CSA on infected erythrocytes. Conclusion Short continuous regions of VAR2CSA with affinity for multiple types of CSA were defined. A number of these regions localize to CSA-binding domains and to surface-exposed regions within these domains and a synthetic peptide corresponding to a peptide sequence in DBL2 was shown to bind to CSA and not to CSC. It is likely that some of these epitopes are involved in native parasite CSA adhesion. However, antibodies directed against single epitopes did not inhibit parasite adhesion. This study supports phage display as a technique to identify CSA-binding regions of large proteins such as VAR2CSA.

Background Resistance of Plasmodium falciparum to atovaquone in vitro and in vivo has been associated to mutations in the parasite cytochrome b gene. Methods Cultures were sequentially subjected to increasing doses of atovaquone alone or in combination with cycloguanil and the cytochrome b gene was sequenced. Additionally, we investigated the parasite cytochrome b gene of a patient returning from Mali with Malarone® treatment failure in vivo. Results All strains that survived atovaquone concentrations in vitro of 2 × 10-8 to 2 × 107 M showed the M133I mutation and one strain with the highest atovaquone concentration the additional mutation L171F. Sequencing of the in vivo treatment failure revealed a point mutation at codon 268 resulting in an amino acid change from tyrosine to serine. Based on the repeated emergence of mutations at codon 268, but no detection of alterations at codon 133 in vivo, we developed a detection method for the diagnostic of codon 268 polymorphisms as a potential atovaquone/proguanil resistance marker. A nested PCR with 3 different pairs of primers for the second round was designed. Each product was digested with restriction enzymes, capable to distinguish the wild type from the two reported mutations at codon 268. Conclusion Mutations at codon 268 of the parasite cytochrome bc1 gene are associated with atovaquone/proguanil treatment failure in vivo and can be used as potential resistance marker This method provides a novel and robust tool to investigate the relevance of codon 268 polymorphisms as resistance marker and to monitor the further emergence of atovaquone/proguanil resistance.

Background Single nucleotide polymorphisms (SNPs) in the Plasmodium vivax dihydrofolate reductase (Pfdhfr) and dihydropteroate synthetase (Pvdhps) genes cause parasite resistance to the antifolate drug combination, sulphadoxine/pyrimethamine (SP). Monitoring these SNPs provide insights into the level of drug pressure caused by SP use and presumably other antifolate drugs. In Sri Lanka, chloroquine (CQ) with primaquine (PQ) and SP with PQ is used as first and second line treatment, respectively, against uncomplicated Plasmodium falciparum and/or P. vivax infections. CQ/PQ is still efficacious against P. vivax infections, thus SP is rarely used and it is assumed that the prevalence of SNPs related to P. vivax SP resistance is low. However, this has not been assessed in Sri Lanka as in most other parts of Asia. This study describes the prevalence and distribution of SNPs related to P. vivax SP resistance across Sri Lanka. Subjects and methods P. vivax- positive samples were collected from subjects presenting at government health facilities across nine of the major malaria endemic districts on the island. The samples were analysed for SNPs/haplotypes at codon 57, 58, 61 and 117 of the Pvdhfr gene and 383, 553 and 585 of the Pvdhps gene by applying PCR followed by a hybridization step using sequence specific oligonucleotide probes (SSOPs) in an ELISA format. Results In the study period, the government of Sri Lanka recorded 2,149 P. vivax cases from the nine districts out of which, 454 (21.1%) blood samples were obtained. Pvdhfr haplotypes could be constructed for 373 of these. The FSTS wild-haplotype was represented in 257 samples (68.9%), the double mutant LRTS haplotype was the most frequently observed mutant (24.4%) while the triple mutation (LRTN) was only identified once. Except for two samples of the single mutated Pvdhps GAV haplotype, the remaining samples were wildtype. Geographical differences were apparent, notably a significantly higher frequency of mutant Pvdhfr haplotypes was observed in the Northern districts. Conclusion Since SP is rarely used in Sri Lanka, the high frequency and diversity of Pvdhfr mutations was unexpected indicating the emergence of drug resistant parasites despite a low level of SP drug pressure.

Background Parasites causing severe malaria in non-immune patients express a restricted subset of variant surface antigens (VSA), which are better recognized by immune sera than VSA expressed during non-severe disease in semi-immune individuals. The most prominent VSA are the var gene-encoded Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family, which is expressed on the surface of infected erythrocytes where it mediates binding to endothelial receptors. Thus, severe malaria may be caused by parasites expressing PfEMP1 variants that afford parasites optimal sequestration in immunologically naïve individuals and high effective multiplication rates. Methods var gene transcription was analysed using real time PCR and PfEMP1 expression by western blots as well as immune plasma recognition of parasite cultures established from non-immune volunteers shortly after infection with NF54 sporozoites. Results In cultures representing the first generation of parasites after hepatic release, all var genes were transcribed, but GroupA var genes were transcribed at the lowest levels. In cultures established from second or third generation blood stage parasites of volunteers with high in vivo parasite multiplication rates, the var gene transcription pattern differed markedly from the transcription pattern of the cultures representing first generation parasites. This indicated that parasites expressing specific var genes, mainly belonging to group A and B, had expanded more effectively in vivo compared to parasites expressing other var genes. The differential expression of PfEMP1 was confirmed at the protein level by immunoblot analysis. In addition, serological typing showed that immune sera more often recognized second and third generation parasites than first generation parasites. Conclusion In conclusion, the results presented here support the hypothesis that parasites causing severe malaria express a subset of PfEMP1, which bestows high parasite growth rates in individuals with limited pre-existing immunity.

Resistance of Plasmodium falciparum to atovaquone in vitro and in vivo has been associated to mutations in the parasite cytochrome b gene. Cultures were sequentially subjected to increasing doses of atovaquone alone or in combination with cycloguanil and the cytochrome b gene was sequenced. Additionally, we investigated the parasite cytochrome b gene of a patient returning from Mali with Malarone® treatment failure in vivo. All strains that survived atovaquone concentrations in vitro of 2 × 10-8 to 2 × 107 M showed the M133I mutation and one strain with the highest atovaquone concentration the additional mutation L171F. Sequencing of the in vivo treatment failure revealed a point mutation at codon 268 resulting in an amino acid change from tyrosine to serine. Based on the repeated emergence of mutations at codon 268, but no detection of alterations at codon 133 in vivo, we developed a detection method for the diagnostic of codon 268 polymorphisms as a potential atovaquone/proguanil resistance marker. A nested PCR with 3 different pairs of primers for the second round was designed. Each product was digested with restriction enzymes, capable to distinguish the wild type from the two reported mutations at codon 268. Mutations at codon 268 of the parasite cytochrome bc1 gene are associated with atovaquone/proguanil treatment failure in vivo and can be used as potential resistance marker This method provides a novel and robust tool to investigate the relevance of codon 268 polymorphisms as resistance marker and to monitor the further emergence of atovaquone/proguanil resistance.

Background: The variant surface antigen family Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP1) is an important target for protective immunity and is implicated in the pathology of malaria through its ability to adhere to host endothelial receptors. The sequence diversity and organization of the 3D7 PfEMP1 repertoire was investigated on the basis of the complete genome sequence. Methods: Using two tree-building methods we analysed the coding and non-coding sequences of 3D7 var and rif genes as well as var genes of other parasite strains. Results: var genes can be sub-grouped into three major groups (group A, B and C) and two intermediate groups B/A and B/C representing transitions between the three major groups. The best defined var group, group A, comprises telomeric genes transcribed towards the telomere encoding PfEMP1s with complex domain structures different from the 4-domain type dominant of groups B and C. Two sequences belonging to the var1 and var2 subfamilies formed independent groups. A rif subgroup transcribed towards the centromere was found neighbouring var genes of group A such that the rif and var 5' regions merged. This organization appeared to be unique for the group A var genes Conclusion: The grouping of var genes implies that var gene recombination preferentially occurs within var gene groups and it is speculated that the groups reflect a functional diversification evolved to cope with the varying conditions of transmission and host immune response met by the parasite.

Background: Malaria, caused by Plasmodium falciparum, continues to have a devastating impact on global health, emphasizing the great need for a malaria vaccine. The circumsporozoite protein (CSP) is an attractive target for a malaria vaccine, and forms a major component of RTS,S, the most clinically advanced malaria vaccine. The clinical efficacy of RTS,S has been moderate, yet has demonstrated the viability of a CSP-based malaria vaccine. In this study, a vaccine comprised of the full-length CSP antigen presented on a virus-like particle (VLP) is produced using a split-intein conjugation system (SpyTag/SpyCatcher) and the immunogenicity is tested in mice.Methods: Full-length 3d7 CSP protein was genetically fused at the C-terminus to SpyCatcher. The CSP-SpyCatcher antigen was then covalently attached (via the SpyTag/SpyCatcher interaction) to Acinetobacter phage AP205 VLPs which were modified to display one SpyTag per VLP subunit. To evaluate the VLP-display effect, the immunogenicity of the VLP vaccine was tested in mice and compared to a control vaccine containing AP205 VLPs plus unconjugated CSP.Results: Full-length CSP was conjugated at high density (an average of 112 CSP molecules per VLP) to AP205 SpyTag-VLPs. Vaccination of mice with the CSP Spy-VLP vaccine resulted in significantly increased antibody titres over a course of 7 months as compared to the control group (2.6-fold higher at 7 months after immunization). Furthermore, the CSP Spy-VLP vaccine appears to stimulate production of IgG2a antibodies, which has been linked with a more efficient clearing of intracellular parasite infection.Conclusion: This study demonstrates that the high-density display of CSP on SpyTag-VLPs, significantly increases the level and quality of the vaccine-induced humoral response, compared to a control vaccine consisting of soluble CSP plus AP205 VLPs. The SpyTag-VLP platform utilized in this study constitutes a versatile and rapid method to develop highly immunogenic vaccines. It might serve as a generic tool for the cost-effective development of effective VLP-vaccines, e.g., against malaria.

Background Antibodies (Ab) to VAR2CSA prevent Plasmodium falciparum-infected erythrocytes from sequestrating in the placenta, i.e., prevent placental malaria (PM). The specificity of Ab to VAR2CSA associated with absence of PM is unknown. Accordingly, differences in the specificity of Ab to VAR2CSA were compared between multigravidae with and without PM who had Ab to VAR2CSA. Methods In a retrospective case–control study, plasma collected from Cameroonian multigravidae with (n = 96) and without (n = 324) PM were screened in 21 assays that measured antibody levels to full length VAR2CSA (FV2), individual VAR2CSA DBL domains, VAR2CSA domains from different genetic backgrounds (variants), as well as proportion of high avidity Ab to FV2. Results Multigravidae with and without PM had similar levels of Ab to FV2, the six VAR2CSA DBL domains and different variants, while the proportion of high avidity Ab to FV2 was significantly higher in women without PM at delivery (p = 0.0030) compared to women with PM. In a logistic regression model adjusted for gravidity and age, the percentage of high avidity Ab to FV2 was associated with reduced likelihood of PM in multigravidae. A 5 % increase in proportion of high avidity Ab to FV2 was associated with a nearly 15 % lower likelihood of PM. Conclusion Ab avidity to FV2 may be an important indicator of immunity to PM. Electronic supplementary material The online version of this article (doi:10.1186/s12936-015-1023-6) contains supplementary material, which is available to authorized users.

Background The insertion of parasite antigens into the host erythrocyte membrane and the structure and distribution of Plasmodium falciparum adhesion receptors on that membrane are poorly understood. Laser scanning confocal microscopy (LSCM) and a novel labelling and fixation method have been used to obtain high resolution immuno-fluorescent images of erythrocyte surface PfEMP1 and internal antigens which allow analysis of the accumulation of PfEMP1 on the erythrocyte membrane during asexual development. Methods A novel staining technique has been developed which permits distinction between erythrocyte surface PfEMP1 and intracellular PfEMP1, in parasites whose nuclear material is exceptionally well resolved. Primary antibody detection by fluorescence is carried out on the live parasitized erythrocyte. The surface labelled cells are then fixed using paraformaldehyde and permeabilized with a non-ionic detergent to permit access of antibodies to internal parasite antigens. Differentiation between surface and internal antigens is achieved using antibodies labelled with different fluorochromes and confocal microscopy Results Surface exposed PfEMP1 is first detectable by antibodies at the trophozoite stage of intracellular parasite development although the improved detection method indicates that there are differences between different laboratory isolates in the kinetics of accumulation of surface-exposed PfEMP1. Conclusion A sensitive method for labelling surface and internal PfEMP1 with up to three different fluorochromes has been developed for laser scanning confocal optical microscopy and the analysis of the developmental expression of malaria adhesion antigens.

Background The var multigene family encodes PfEMP1, which are expressed on the surface of infected erythrocytes and bind to various host endothelial receptors. Antigenic variation of PfEMP1 plays a key role in malaria pathogenesis, a process partially controlled at the level of var gene transcription. Transcriptional levels, throughout the intra-erythrocytic cycle, of 59 var genes of the NF54 clone were measured simultaneously by quantitative real-time PCR. The timing of var transcript abundance, the number of genes transcribed and whether transcripts were correctly spliced for protein expression were determined. Two parasite populations were studied; an unselected population of NF54 and a selected population, NF54VAR2CSA, to compare both the transcription of var2csa and the expression pattern of the corresponding protein. Methods Synchronized parasites were harvested at different time points along the 48 hours intra-erythrocytic cycle for extraction of RNA and for analysis of expression of variant surface antigens by flow cytometry. Total RNA from each parasite sample was extracted and cDNA synthesized. Quantitative real-time PCR was performed using gene-specific primers for all var genes. Samples for flow cytometry were labelled with rabbit IgG targeting DBL5ε of VAR2CSA and serum IgG from malaria-exposed men and pregnant women. Results var transcripts were detected at all time points of the intra-erythrocytic cycle by quantitative real-time PCR, although transcription peaked in ring-stage parasites. There was no difference in the timing of appearance of group A, B or C transcripts, and dominant and subdominant var transcripts appeared to be correctly spliced at all time points. VAR2CSA appeared on the surface of infected erythrocytes 16 hours after invasion, consistent with previous studies of other PfEMP1. Transcription of the pseudogene var1csa could not be detected in NF54VAR2CSA cells. Conclusion The optimal sampling point for analysis of var transcripts using quantitative real-time PCR is the ring-stage, which is encouraging for the analysis of fresh clinical isolates. The data presented here indicate that there is no promiscuous transcription of var genes at the individual cell level and that it is possible to correlate dominant transcripts with adhesion phenotype and clinical markers of malaria severity.

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 DBL5ε- 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- DBL5ε- 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.