Plasmodium falciparum malaria remains one of the world's foremost health problems, primarily in highly endemic regions such as Sub-Saharan Africa, where it is responsible for substantial morbidity, mortality and economic losses. Malaria is a significant cause of severe disease and death in pregnant women and newborns, with pathogenesis being associated with expression of a unique variant of the multidomain Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) called VAR2CSA. Here, we characterize the polymorphism of the DBL3X domain of VAR2CSA and identify regions under selective pressure among placental parasites from women living in endemic western Kenya. In addition to significant levels of polymorphism, our analysis reveals evidence for diversification through intra-segmental recombination and novel mutations that likely contributed to the high number of unique VAR2CSA sequence types identified in this study. Interestingly, we also identified a number of critical residues that may be implicated in immune evasion through switching (or toggling) to alternative amino acids, including an arginine residue within the predicted binding pocket in subdomain III, which was previously implicated in binding to placental CSA. Overall, these findings are important for understanding parasite diversity in pregnant women and will be useful for identifying epitopes and variants of DBL3X to be included in a vaccine against placental malaria.

Placental malaria, caused by sequestration of Plasmodium falciparum-infected erythrocytes in the placenta, is associated with increased risk of maternal morbidity and poor birth outcomes. The parasite antigen VAR2CSA (variant surface antigen 2-chondroitin sulfate A) is expressed on infected erythrocytes and mediates binding to chondroitin sulfate A, initiating inflammation and disrupting homeostasis at the maternal-fetal interface. Although antibodies can prevent sequestration, it is unclear whether parasite clearance is due to antibodies to a single Duffy binding-like (DBL) domain or to an extensive repertoire of antibodies to multiple DBL domains and allelic variants. Accordingly, plasma samples collected longitudinally from pregnant women were screened for naturally acquired antibodies against an extensive panel of VAR2CSA proteins, including 2 to 3 allelic variants for each of 5 different DBL domains. Analyses were performed on plasma samples collected from 3 to 9 months of pregnancy from women living in areas in Cameroon with high and low malaria transmission. The results demonstrate that high antibody levels to multiple VAR2CSA domains, rather than a single domain, were associated with the absence of placental malaria when antibodies were present from early in the second trimester until term. Absence of placental malaria was associated with increasing antibody breadth to different DBL domains and allelic variants in multigravid women. Furthermore, the antibody responses of women in the lower-transmission site had both lower magnitude and lesser breadth than those in the high-transmission site. These data suggest that immunity to placental malaria results from high antibody levels to multiple VAR2CSA domains and allelic variants and that antibody breadth is influenced by malaria transmission intensity.

Var2CSA, a key molecule linked with pregnancy-associated malaria (PAM), causes sequestration of Plasmodium falciparum infected erythrocytes (PEs) in the placenta by adhesion to chondroitin sulfate A (CSA). Var2CSA possesses a 300 kDa extracellular region composed of six Duffy-binding like (DBL) domains and a cysteine-rich interdomain region (CIDRpam) module. Although initial studies implicated several individual var2CSA DBL domains as important for adhesion of PEs to CSA, new studies revealed that these individual domains lack both the affinity and specificity displayed by the full-length extracellular region. Indeed, recent evidence suggests the presence of a single CSA-binding site formed by a higher-order domain organization rather than several independent binding sites located on the different domains. Here, we search for the minimal binding region within var2CSA that maintains high affinity and specificity for CSA binding, a characteristic feature of the full-length extracellular region. Accordingly, truncated recombinant var2CSA proteins comprising different domain combinations were expressed and their binding characteristics assessed against different sulfated glycosaminoglycans (GAGs). Our results indicate that the smallest region within var2CSA with similar binding properties to those of the full-length var2CSA is DBL1X-3X. We also demonstrate that inhibitory antibodies raised in rabbit against the full-length DBL1X-6ε target principally DBL3X and, to a lesser extent, DBL5ε. Taken together, our results indicate that efforts should focus on the DBL1X-3X region for developing vaccine and therapeutic strategies aimed at combating PAM.

Malaria during pregnancy is a major health problem for African women. The disease is caused by Plasmodium falciparum malaria parasites, which accumulate in the placenta by adhering to chondroitin sulfate A (CSA). The interaction between infected erythrocytes and the placental receptor is mediated by a parasite expressed protein named VAR2CSA. A vaccine protecting pregnant women against placental malaria should induce antibodies inhibiting the interaction between VAR2CSA and CSA. Much effort has been put into defining the part of the 350 kDa VAR2CSA protein that is responsible for binding. It has been shown that full-length recombinant VAR2CSA binds specifically to CSA with high affinity, however to date no sub-fragment of VAR2CSA has been shown to interact with CSA with similar affinity or specificity. In this study, we used a biosensor technology to examine the binding properties of a panel of truncated VAR2CSA proteins. The experiments indicate that the core of the CSA-binding site is situated in three domains, DBL2X-CIDR(PAM) and a flanking domain, located in the N-terminal part of VAR2CSA. Furthermore, recombinant VAR2CSA subfragments containing this region elicit antibodies with high parasite adhesion blocking activity in animal immunization experiments.

The high molecular weight, multidomain VAR2CSA protein mediating adhesion of Plasmodium falciparum-infected erythrocytes in the placenta is the leading candidate for a pregnancy malaria vaccine. However, it has been difficult so far to generate strong and consistent adhesion blocking antibody responses against most single-domain VAR2CSA immunogens. Recent advances in expression of the full-length recombinant protein showed it binds with much greater specificity and affinity to chondroitin sulphate A (CSA) than individual VAR2CSA domains. This raises the possibility that a specific CSA binding pocket(s) is formed in the full length antigen and could be an important target for vaccine development. In this study, we compared the immunogenicity of a full-length VAR2CSA recombinant protein containing all six Duffy binding-like (DBL) domains to that of a three-domain construct (DBL4-6) in mice and rabbits. Animals immunized with either immunogen acquired antibodies reacting with several VAR2CSA individual domains by ELISA, but antibody responses against the highly conserved DBL4 domain were weaker in animals immunized with full-length DBL1-6 recombinant protein compared to DBL4-6 recombinant protein. Both immunogens induced cross-reactive antibodies to several heterologous CSA-binding parasite lines expressing different VAR2CSA orthologues. However, antibodies that inhibited adhesion of parasites to CSA were only elicited in rabbits immunized with full-length immunogen and inhibition was restricted to the homologous CSA-binding parasite. These findings demonstrate that partial and full-length VAR2CSA immunogens induce cross-reactive antibodies, but inhibitory antibody responses to full-length immunogen were highly allele-specific and variable between animal species.

Pregnancy associated malaria is a severe clinical syndrome associated with sequestration of Plasmodium falciparum-infected erythrocytes in the placenta. Placental binding is mediated by VAR2CSA, which adheres to chondroitin sulphate A (CSA). VAR2CSA is a large and polymorphic protein that has six Duffy binding-like (DBL) domains. There is still limited understanding as to how effective individual VAR2CSA domains are at generating inhibitory antibodies or the number of domain variants needed for universal vaccine coverage. To investigate the immunogenic properties of single domain VAR2CSA recombinant proteins, rats or rabbits were immunized with five of the six VAR2CSA domains produced in Pichia pastoris. Immune plasma was analysed against a geographically diverse panel of CSA-binding lab lines to assess antibody breadth and inhibitory activity. Of the five domains, DBL3, and to a lesser extent DBL5, induced antibodies that cross-reacted on five diverse CSA-binding parasite lines by flow cytometry. By comparison, anti-DBL6 antibodies were highly strain-specific and anti-DBL1 and anti-DBL4 antibodies were poorly reactive by flow cytometry. From this series of recombinant proteins, adhesion-blocking activity was restricted to a single rat immunized against a DBL4 recombinant protein. Single domain VAR2CSA recombinant proteins produced in P. pastoris had limited efficacy in eliciting adhesion blocking antibody responses, but VAR2CSA DBL3 and DBL5 domains contain strain-transcendent epitopes that can be targeted by vaccination and may have application for vaccine development.

Acquired protection from Plasmodium falciparum placental malaria, a major cause of maternal, fetal, and infant morbidity, is mediated by IgG specific for the P. falciparum erythrocyte membrane protein 1 variant VAR2CSA. This protein enables adhesion of P. falciparum-infected erythrocytes to chondroitin sulfate A in the intervillous space. Although interclonal variation of the var2csa gene is lower than that among var genes in general, VAR2CSA-specific Abs appear to target mainly polymorphic epitopes. This has raised doubts about the feasibility of VAR2CSA-based vaccines. We used eight human monoclonal IgG Abs from affinity-matured memory B cells of P. falciparum-exposed women to study interclonal variation and functional importance of Ab epitopes among placental and peripheral parasites from East and West Africa. Most placental P. falciparum isolates were labeled by several mAbs, whereas peripheral isolates from children were essentially nonreactive. The mAb reactivity of peripheral isolates from pregnant women indicated that some were placental, whereas others had alternative sequestration foci. Most of the mAbs were comparable in their reactivity with bound infected erythrocytes (IEs) and recombinant VAR2CSA and interfered with IE and/or VAR2CSA binding to chondroitin sulfate A. Pair-wise mAb combinations were more inhibitory than single mAbs, and all of the mAbs together was the most efficient combination. Each mAb could opsonize IEs for phagocytosis, and a combination of the eight mAbs caused phagocytosis similar to that of plasma IgG-opsonized IEs. We conclude that functionally important Ab epitopes are shared by the majority of polymorphic VAR2CSA variants, which supports the feasibility of VAR2CSA-based vaccines against placental malaria.

Interactions between parasite-encoded proteins and host carbohydrate molecules are essential at multiple stages in the life cycle of the malaria-causing parasite, Plasmodium falciparum, and these interactions are targets for the development of therapeutics to treat the disease. Here we review recent structural studies of carbohydrate binding modules that mediate recognition events important for cell invasion and cytoadhesion. In particular we focus on the structures of two molecules; the erythrocyte binding antigen, EBA-175 involved in erythrocyte invasion and the VAR2CSA protein that mediates binding of infected erythrocytes to the placenta. These proteins both use Duffy-binding like (DBL) domains, a Plasmodium specific fold, to bind host carbohydrates, but recent results show that they differ significantly in their architectures and modes of ligand recognition.

Mathematical models of malaria transmission have been used to inform the design of malaria control programs since the mid 20th century, and many of these models have provided useful insights into the complexity of the disease. Among developing countries, however and particularly in sub-Saharan Africa, malaria remains a major cause of morbidity and mortality. One of the main difficulties in controlling the most virulent human malaria parasite, Plasmodium falciparum, is its genetic diversity, which confounds attempts to design an effective vaccine. The population structure of P. falciparum remains poorly understood but plays a key role in determining epidemiological patterns of disease and the development of immunity. We discuss the seminal model of malaria transmission developed by Ross and MacDonald, and the modifications that have been made since to include more realism. We show that age profiles of disease and serological data support a theoretical model in which the parasite population is diverse and structured into several antigenic types and highlight the implications of this structure for controlling malaria. Lastly, we discuss the current sequence data on parasite antigen genes that are important for the aquisition of immunity, and the results of a new analysis of P. falciparum population structure at the genomic level.

Pregnancy-associated malaria (PAM) is a serious consequence of sequestration of Plasmodium falciparum-parasitized erythrocytes (PE) in the placenta through adhesion to chondroitin sulfate A (CSA) present on placental proteoglycans. Recent work implicates var2CSA, a member of the PfEMP1 family, as the mediator of placental sequestration and as a key target for PAM vaccine development. Var2CSA is a 350 kDa transmembrane protein, whose extracellular region includes six Duffy-binding-like (DBL) domains. Due to its size and high cysteine content, the full-length var2CSA extracellular region has not hitherto been expressed in heterologous systems, thus limiting investigations to individual recombinant domains. Here we report for the first time the expression of the full-length var2CSA extracellular region (domains DBL1X to DBL6epsilon) from the 3D7 parasite strain using the human embryonic kidney 293 cell line. We show that the recombinant extracellular var2CSA region is correctly folded and that, unlike the individual DBL domains, it binds with high affinity and specificity to CSA (K(D)= 61 nM) and efficiently inhibits PE from binding to CSA. Structural characterization by analytical ultracentrifugation and small-angle x-ray scattering reveals a compact organization of the full-length protein, most likely governed by specific interdomain interactions, rather than an extended structure. Collectively, these data suggest that a high-affinity, CSA-specific binding site is formed by the higher-order structure of the var2CSA extracellular region. These results have important consequences for the development of an effective vaccine and therapeutic inhibitors.

In Plasmodium falciparum, var genes encode adhesive proteins that are transported to the surface of infected erythrocytes and act as major virulence determinants for infected erythrocyte binding and immune evasion. Var genes are highly diverse and can be classified into five major groups (UpsA, B, C, D, and E). Previous serological studies have suggested that the UpsA var group may contain common antigenic types that have important roles in severe childhood malaria. Here, our analysis found that UpsA vars are highly diverse between 22 world-wide parasite isolates, although they could be grouped into two broad clusters that may be separately recombining. By comparison, orthologs of the UpsA-linked Type 3 var and UpsE-linked var2csa were detected in nearly all parasite isolates, and a var2csa ortholog was also present in a chimpanzee malaria P. reichenowi that diverged from P. falciparum approximately 5-7 million years ago. Although the specific function of Type 3 var genes is unknown, var2csa is a leading candidate for a pregnancy associated malaria vaccine. Compared to typical var genes, var2csa is unusually conserved but still had only 54-94% amino acid identity in extracellular binding regions. However, var2csa alleles have extensive gene mosaicism within polymorphic blocks that are shared between world-wide parasite isolates and recognizable in P. rechenowi suggesting a high rate of self-self recombination and an ancient and globally-related pool of var2csa polymorphism. These studies aid our understanding of the evolutionary mechanisms that shape var diversity and will be important to the development of vaccines against pregnancy associated malaria and severe malaria.

Cerebral malaria (CM) is a deadly complication of Plasmodium falciparum infection, but specific interactions involved in cerebral homing of infected erythrocytes (IEs) are poorly understood. In this study, P. falciparum-IEs were characterized for binding to primary human brain microvascular endothelial cells (HBMECs). Before selection, CD36 or ICAM-1-binding parasites exhibited punctate binding to a subpopulation of HBMECs and binding was CD36 dependent. Panning of IEs on HBMECs led to a more dispersed binding phenotype and the selection of three var genes, including two that encode the tandem domain cassette 8 (DC8) and were non-CD36 binders. Multiple domains in the DC8 cassette bound to brain endothelium and the cysteine-rich interdomain region 1 inhibited binding of P. falciparum-IEs by 50%, highlighting a key role for the DC8 cassette in cerebral binding. It is mysterious how deadly binding variants are maintained in the parasite population. Clonal parasite lines expressing the two brain-adherent DC8-var genes did not bind to any of the known microvascular receptors, indicating unique receptors are involved in cerebral binding. They could also adhere to brain, lung, dermis, and heart endothelial cells, suggesting cerebral binding variants may have alternative sequestration sites. Furthermore, young African children with CM or nonsevere control cases had antibodies to HBMEC-selected parasites, indicating they had been exposed to related variants during childhood infections. This analysis shows that specific P. falciparum erythrocyte membrane protein 1 types are linked to cerebral binding and suggests a potential mechanism by which individuals may build up immunity to severe disease, in the absence of CM.

The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family plays a central role in antigenic variation and cytoadhesion of P. falciparum infected erythrocytes. PfEMP1 proteins/var genes are classified into three main subfamilies (UpsA, UpsB, and UpsC) that are hypothesized to have different roles in binding and disease. To investigate whether these subfamilies have diverged in binding specificity and test if binding could be predicted by adhesion domain classification, we generated a panel of 19 parasite lines that primarily expressed a single dominant var transcript and assayed binding against 12 known host receptors. By limited dilution cloning, only UpsB and UpsC var genes were isolated, indicating that UpsA var gene expression is rare under in vitro culture conditions. Consequently, three UpsA variants were obtained by rosette purification and selection with specific monoclonal antibodies to create a more representative panel. Binding assays showed that CD36 was the most common adhesion partner of the parasite panel, followed by ICAM-1 and TSP-1, and that CD36 and ICAM-1 binding variants were highly predicted by adhesion domain sequence classification. Binding to other host receptors, including CSA, VCAM-1, HABP1, CD31/PECAM, E-selectin, Endoglin, CHO receptor "X", and Fractalkine, was rare or absent. Our findings identify a category of larger PfEMP1 proteins that are under dual selection for ICAM-1 and CD36 binding. They also support that the UpsA group, in contrast to UpsB and UpsC var genes, has diverged from binding to the major microvasculature receptor CD36 and likely uses other mechanisms to sequester in the microvasculature. These results demonstrate that CD36 and ICAM-1 have left strong signatures of selection on the PfEMP1 family that can be detected by adhesion domain sequence classification and have implications for how this family of proteins is specializing to exploit hosts with varying levels of anti-malaria immunity.

ABSTRACT: BACKGROUND: The human malaria parasite Plasmodium falciparum survives pressures from the host immune system and antimalarial drugs by modifying its genome. Genetic recombination and nucleotide substitution are the two major mechanisms that the parasite employs to generate genome diversity. A better understanding of these mechanisms may provide important information for studying parasite evolution, immune evasion and drug resistance. RESULTS: Here, we used a high-density tiling array to estimate the genetic recombination rate among 32 progeny of a P. falciparum genetic cross (7G8 × GB4). We detected 638 recombination events and constructed a high-resolution genetic map. Comparing genetic and physical maps, we obtained an overall recombination rate of 9.6 kb per centimorgan and identified 54 candidate recombination hotspots. Similar to centromeres in other organisms, the sequences of P. falciparum centromeres are found in chromosome regions largely devoid of recombination activity. Motifs enriched in hotspots were also identified, including a 12-bp G/C-rich motif with 3-bp periodicity that may interact with a protein containing 11 predicted zinc finger arrays. CONCLUSIONS: These results show that the P. falciparum genome has a high recombination rate, although it also follows the overall rule of meiosis in eukaryotes with an average of approximately one crossover per chromosome per meiosis. GC-rich repetitive motifs identified in the hotspot sequences may play a role in the high recombination rate observed. The lack of recombination activity in centromeric regions is consistent with the observations of reduced recombination near the centromeres of other organisms.

Deep resequencing of functional regions in human genomes is key to identifying potentially causal rare variants for complex disorders. Here, we present the results from a large-sample resequencing (n  =  285 patients) study of candidate genes coupled with population genetics and statistical methods to identify rare variants associated with Autism Spectrum Disorder and Schizophrenia. Three genes, MAP1A, GRIN2B, and CACNA1F, were consistently identified by different methods as having significant excess of rare missense mutations in either one or both disease cohorts. In a broader context, we also found that the overall site frequency spectrum of variation in these cases is best explained by population models of both selection and complex demography rather than neutral models or models accounting for complex demography alone. Mutations in the three disease-associated genes explained much of the difference in the overall site frequency spectrum among the cases versus controls. This study demonstrates that genes associated with complex disorders can be mapped using resequencing and analytical methods with sample sizes far smaller than those required by genome-wide association studies. Additionally, our findings support the hypothesis that rare mutations account for a proportion of the phenotypic variance of these complex disorders.

The role of de novo mutations (DNMs) in common diseases remains largely unknown. Nonetheless, the rate of de novo deleterious mutations and the strength of selection against de novo mutations are critical to understanding the genetic architecture of a disease. Discovery of high-impact DNMs requires substantial high-resolution interrogation of partial or complete genomes of families via resequencing. We hypothesized that deleterious DNMs may play a role in cases of autism spectrum disorders (ASD) and schizophrenia (SCZ), two etiologically heterogeneous disorders with significantly reduced reproductive fitness. We present a direct measure of the de novo mutation rate (μ) and selective constraints from DNMs estimated from a deep resequencing data set generated from a large cohort of ASD and SCZ cases (n = 285) and population control individuals (n = 285) with available parental DNA. A survey of ∼430 Mb of DNA from 401 synapse-expressed genes across all cases and 25 Mb of DNA in controls found 28 candidate DNMs, 13 of which were cell line artifacts. Our calculated direct neutral mutation rate (1.36 × 10(-8)) is similar to previous indirect estimates, but we observed a significant excess of potentially deleterious DNMs in ASD and SCZ individuals. Our results emphasize the importance of DNMs as genetic mechanisms in ASD and SCZ and the limitations of using DNA from archived cell lines to identify functional variants.

Pregnant women are infected by specific variants of Plasmodium falciparum that adhere and accumulate in the placenta. Using serological and molecular approaches, we assessed the global antigenic diversity of surface antigens expressed by placental-binding isolates to better understand immunity to malaria in pregnancy and evolution of polymorphisms, and to inform vaccine development. We found that placental-binding isolates originating from all major regions where malaria occurs were commonly recognised by antibodies among different populations of pregnant women. There was substantial antigenic overlap and sharing of epitopes between isolates, including those from distant geographic locations, suggesting limitations to antigenic diversity; however, differences between populations and isolates were also seen. Many women had cross-reactive antibodies and/or a broad repertoire of antibodies to different isolates. Studying VAR2CSA as the major antigen expressed by placental-binding isolates, we identified antibody epitopes encoded by variable sequence blocks in the DBL3 domain. Analysis of global var2csa DBL3 sequences demonstrated extensive sharing of variable blocks between Africa, Asia, PNG, and Latin America, which likely contributes to the high level of antigenic overlap between different isolates. However, there was also evidence of geographic clustering of sequences and differences in VAR2CSA sequences between populations. Results point to limited antigenic diversity of placental-binding isolates and may explain why immunity to malaria in pregnancy can be achieved after exposure during one pregnancy. The inclusion of a limited number of variants in a candidate vaccine may be sufficient for broad population coverage, but geographic considerations may be also needed in vaccine design.

Antimalarial drugs impose strong selective pressure on Plasmodium falciparum parasites and leave signatures of selection in the parasite genome; screening for genes under selection may suggest potential drug or immune targets. Genome-wide association studies (GWAS) of parasite traits have been hampered by the lack of high-throughput genotyping methods, inadequate knowledge of parasite population history and time-consuming adaptations of parasites to in vitro culture. Here we report the first Plasmodium GWAS, which included 189 culture-adapted P. falciparum parasites genotyped using a custom-built Affymetrix molecular inversion probe 3K malaria panel array with a coverage of approximately 1 SNP per 7 kb. Population structure, variation in recombination rate and loci under recent positive selection were detected. Parasite half-maximum inhibitory concentrations for seven antimalarial drugs were obtained and used in GWAS to identify genes associated with drug responses. This study provides valuable tools and insight into the P. falciparum genome.

BACKGROUND Malaria kills more people worldwide than all inherited human genetic disorders combined. To characterize how the parasites causing this disease adapt to different host environments, we compared the evolutionary genomics of two distinct groups of malaria pathogens in order to identify critical properties associated with infection of different hosts: those parasites infecting hominids (Plasmodium falciparum and P. reichenowi) versus parasites infecting rodent hosts (P. yoelii yoelii, P. berghei, and P. chabaudi). Adaptation by the parasite to its host is likely highly critical to the evolution of these species. RESULTS Our comparative analysis suggests that patterns of molecular evolution in the hominid parasite lineage are generally similar to those of the rodent lineage but distinct in several aspects. The most rapidly evolving genes in both lineages are those involved in host-parasite interactions as well as those that show the lowest expression levels. However, we found that, similar to their respective mammal host lineages, parasite genomes infecting hominids are generally less constrained, evolving at faster rates, and accumulating more deleterious mutations than those infecting murids, which may reflect an historical lower effective size of the hominid lineage and relaxed host-driven selective pressures. CONCLUSION Our study highlights for the first time the differences in trends and rates of evolution in Plasmodium lineages infecting different hosts and emphasizes the potential importance of the variation in effective size between lineages to explain variation in selective constraints among genomes.

Infection with Plasmodium falciparum during pregnancy is one of the major causes of malaria related morbidity and mortality in newborn and mothers. The complications of pregnancy-associated malaria result mainly from massive adhesion of Plasmodium falciparum-infected erythrocytes (IE) to chondroitin sulfate A (CSA) present in the placental intervillous blood spaces. Var2CSA, a member of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family is the predominant parasite ligand mediating CSA binding. However, experimental evidence suggests that other host receptors, such as hyaluronic acid (HA) and the neonatal Fc receptor, may also support placental binding. Here we used parasites in which var2csa was genetically disrupted to evaluate the contribution of these receptors to placental sequestration and to identify additional adhesion receptors that may be involved in pregnancy-associated malaria. By comparison to the wild-type parasites, the FCR3Deltavar2csa mutants could not be selected for HA adhesion, indicating that var2csa is not only essential for IE cytoadhesion to the placental receptor CSA, but also to HA. However, further studies using different pure sources of HA revealed that the previously observed binding results from CSA contamination in the bovine vitreous humor HA preparation. To identify CSA-independent placental interactions, FCR3Deltavar2csa mutant parasites were selected for adhesion to the human placental trophoblastic BeWo cell line. BeWo selected parasites revealed a multi-phenotypic adhesion population expressing multiple var genes. However, these parasites did not cytoadhere specifically to the syncytiotrophoblast lining of placental cryosections and were not recognized by sera from malaria-exposed women in a parity dependent manner, indicating that the surface molecules present on the surface of the BeWo selected population are not specifically expressed during the course of pregnancy-associated malaria. Taken together, these results demonstrate that the placental malaria associated phenotype can not be restored in FCR3Deltavar2csa mutant parasites and highlight the key role of var2CSA in pregnancy malaria pathogenesis and for vaccine development.

Acquired immunity in vertebrates maintains polymorphisms in endemic pathogens, leading to identifiable signatures of balancing selection. To comprehensively survey for genes under such selection in the human malaria parasite Plasmodium falciparum, we generated paired-end short-read sequences of parasites in clinical isolates from an endemic Gambian population, which were mapped to the 3D7 strain reference genome to yield high-quality genome-wide coding sequence data for 65 isolates. A minority of genes did not map reliably, including the hypervariable var, rifin, and stevor families, but 5,056 genes (90.9% of all in the genome) had >70% sequence coverage with minimum read depth of 5 for at least 50 isolates, of which 2,853 genes contained 3 or more single nucleotide polymorphisms (SNPs) for analysis of polymorphic site frequency spectra. Against an overall background of negatively skewed frequencies, as expected from historical population expansion combined with purifying selection, the outlying minority of genes with signatures indicating exceptionally intermediate frequencies were identified. Comparing genes with different stage-specificity, such signatures were most common in those with peak expression at the merozoite stage that invades erythrocytes. Members of clag, PfMC-2TM, surfin, and msp3-like gene families were highly represented, the strongest signature being in the msp3-like gene PF10_0355. Analysis of msp3-like transcripts in 45 clinical and 11 laboratory adapted isolates grown to merozoite-containing schizont stages revealed surprisingly low expression of PF10_0355. In diverse clonal parasite lines the protein product was expressed in a minority of mature schizonts (<1% in most lines and ∼10% in clone HB3), and eight sub-clones of HB3 cultured separately had an intermediate spectrum of positive frequencies (0.9 to 7.5%), indicating phase variable expression of this polymorphic antigen. This and other identified targets of balancing selection are now prioritized for functional study.

Signatures of balancing selection operating on specific gene loci in endemic pathogens can identify candidate targets of naturally acquired immunity. In malaria parasites, several leading vaccine candidates convincingly show such signatures when subjected to several tests of neutrality, but the discovery of new targets affected by selection to a similar extent has been slow. A small minority of all genes are under such selection, as indicated by a recent study of 26 Plasmodium falciparum merozoite-stage genes that were not previously prioritized as vaccine candidates, of which only one (locus PF10_0348) showed a strong signature. Therefore, to focus discovery efforts on genes that are polymorphic, we scanned all available shotgun genome sequence data from laboratory lines of P. falciparum and chose six loci with more than five single nucleotide polymorphisms per kilobase (including PF10_0348) for in-depth frequency-based analyses in a Kenyan population (allele sample sizes >50 for each locus) and comparison of Hudson-Kreitman-Aguade (HKA) ratios of population diversity (π) to interspecific divergence (K) from the chimpanzee parasite Plasmodium reichenowi. Three of these (the msp3/6-like genes PF10_0348 and PF10_0355 and the surf(4.1) gene PFD1160w) showed exceptionally high positive values of Tajima's D and Fu and Li's F indices and have the highest HKA ratios, indicating that they are under balancing selection and should be prioritized for studies of their protein products as candidate targets of immunity. Combined with earlier results, there is now strong evidence that high HKA ratio (as well as the frequency-independent ratio of Watterson's /K) is predictive of high values of Tajima's D. Thus, the former offers value for use in genome-wide screening when numbers of genome sequences within a species are low or in combination with Tajima's D as a 2D test on large population genomic samples.

Pregnancy associated malaria is a severe clinical syndrome associated with sequestration of Plasmodium falciparum-infected erythrocytes in the placenta. Placental binding is mediated by VAR2CSA, a member of the large and diverse P. falciparum erythrocyte membrane 1 (PfEMP1) protein family. To better understand if conserved regions in VAR2CSA can be targeted by antibodies, we immunized rabbits with VAR2CSA DBL1 and DBL5 recombinant proteins produced in Pichia pastoris and developed a panel of seven CSA-binding parasites from diverse geographic origins. Overall, no two parasites in the panel expressed the same VAR2CSA sequence. The DBL1 domains averaged 80% amino acid identity (range 72-89%) and the DBL5 domains averaged 86% amino acid identity (range 83-99%), similar to a broader sampling of VAR2CSA sequences from around the world. Whereas antibodies generated against the VAR2CSA DBL1 recombinant protein had only limited breadth and reacted with three or four parasites in the panel, immunization with DBL5 recombinant proteins elicited broadly cross-reactive antibodies against all or most parasites in the panel, as well as to fresh clinical isolates from pregnant women. These findings demonstrate that the major PfEMP1 variant expressed by placental isolates exposes strain-transcendent epitopes that can be targeted by vaccination and may have application for pregnancy malaria vaccine development.

We have identified new malaria vaccine candidates through the combination of bioinformatics prediction of stable protein domains in the Plasmodium falciparum genome, chemical synthesis of polypeptides, in vitro biological functional assays, and association of an antigen-specific antibody response with protection against clinical malaria. Within the predicted open reading frame of P. falciparum hypothetical protein PFF0165c, several segments with low hydrophobic amino acid content, which are likely to be intrinsically unstructured, were identified. The synthetic peptide corresponding to one such segment (P27A) was well recognized by sera and peripheral blood mononuclear cells of adults living in different regions where malaria is endemic. High antibody titers were induced in different strains of mice and in rabbits immunized with the polypeptide formulated with different adjuvants. These antibodies recognized native epitopes in P. falciparum-infected erythrocytes, formed distinct bands in Western blots, and were inhibitory in an in vitro antibody-dependent cellular inhibition parasite-growth assay. The immunological properties of P27A, together with its low polymorphism and association with clinical protection from malaria in humans, warrant its further development as a malaria vaccine candidate.

BACKGROUND The function of the 19 kDa C-terminal region of the merozoite surface protein 1 (MSP1-19) expressed by Plasmodium has been demonstrated to be conserved across distantly related Plasmodium species. The green fluorescent protein (GFP) is a reporter protein that has been widely used because it can be easily detected in living organisms by fluorescence microscopy and flow cytometry. METHODOLOGY AND RESULTS In this study, we used gene targeting to generate transgenic P. berghei (Pb) parasites (designated as PfMSP1-19Pb) that express the MSP1-19 of P. falciparum (Pf) and the GFP reporter protein simultaneously. The replacement of the PbMSP1-19 locus by PfMSP1-19 was verified by PCR and Southern analysis. The expression of the chimeric PbfMSP-1 and the GFP was verified by Western blot and fluorescence microscopy, respectively. Moreover, GFP-expressing transgenic parasites in blood stages can be readily differentiated from other blood cells using flow cytometry. A comparison of growth rates between wild-type and the PfMSP1-19Pb transgenic parasite indicated that the replacement of the MSP1-19 region and the expression of the GFP protein were not deleterious to the transgenic parasites. We used this transgenic mouse parasite as a murine model to evaluate the protective efficacy in vivo of specific IgG elicited by a PfCP-2.9 malaria vaccine that contains the PfMSP1-19. The BALB/c mice passively transferred with purified rabbit IgG to the PfCP-2.9 survived a lethal challenge of the PfMSP1-19Pb transgenic murine parasites, but not the wild-type P. berghei whereas the control mice passively transferred with purified IgG obtained from adjuvant only-immunized rabbits were vulnerable to both transgenic and wild-type infections. CONCLUSIONS We generated a transgenic P. berghei line that expresses PfMSP1-19 and the GFP reporter gene simultaneously. The availability of this parasite line provides a murine model to evaluate the protective efficacy in vivo of anti-MSP1-19 antibodies, including, potentially, those elicited by the PfCP-2.9 malaria vaccine in human volunteers.

BACKGROUND The var2csa gene encodes a Plasmodium falciparum adhesion receptor which binds chondroitin sulfate A (CSA). This var gene is more conserved than other PfEMP1/var genes and is found in all P. falciparum isolates. In isolates 3D7, FCR3/It4 and HB3, var2csa is transcribed from a sub-telomeric position on the left arm of chromosome 12, but it is not known if this location is conserved in all parasites. Genome sequencing indicates that the var2csa gene is duplicated in HB3, but whether this is true in natural populations is uncertain. METHODOLOGY/PRINCIPAL FINDINGS To assess global variation in the VAR2CSA protein, sequence variation in the DBL2X region of var2csa genes in 54 P.falciparum samples was analyzed. Chromosome mapping of var2csa loci was carried out and a quantitative PCR assay was developed to estimate the number of var2csa genes in P.falciparum isolates from the placenta of pregnant women and from the peripheral circulation of other malaria patients. Sequence analysis, gene mapping and copy number quantitation in P.falciparum isolates indicate that there are at least two loci and that both var2csa-like genes can be transcribed. All VAR2CSA DBL2X domains fall into one of two distinct phylogenetic groups possessing one or the other variant of a large (approximately 26 amino acid) dimorphic motif, but whether either motif variant is linked to a specific locus is not known. CONCLUSIONS/SIGNIFICANCE Two or more related but distinct var2csa-type PfEMP1/var genes exist in many P. falciparum isolates. One gene is on chromosome 12 but additional var2csa-type genes are on different chromosomes in different isolates. Multiplicity of var2csa genes appears more common in infected placentae than in samples from non-pregnant donors indicating a possible advantage of this genotype in pregnancy associated malaria.

Falciparum malaria is initiated when Anopheles mosquitoes transmit the Plasmodium sporozoite stage during a blood meal. Irradiated sporozoites confer sterile protection against subsequent malaria infection in animal models and humans. This level of protection is unmatched by current recombinant malaria vaccines. However, the live-attenuated vaccine approach faces formidable obstacles, including development of accurate, reproducible attenuation techniques. We tested whether Plasmodium falciparum could be attenuated at the early liver stage by genetic engineering. The P. falciparum genetically attenuated parasites (GAPs) harbor individual deletions or simultaneous deletions of the sporozoite-expressed genes P52 and P36. Gene deletions were done by double-cross-over recombination to avoid genetic reversion of the knockout parasites. The gene deletions did not affect parasite replication throughout the erythrocytic cycle, gametocyte production, mosquito infections, and sporozoite production rates. However, the deletions caused parasite developmental arrest during hepatocyte infection. The double-gene deletion line exhibited a more severe intrahepatocytic growth defect compared with the single-gene deletion lines, and it did not persist. This defect was assessed in an in vitro liver-stage growth assay and in a chimeric mouse model harboring human hepatocytes. The strong phenotype of the double knockout GAP justifies its human testing as a whole-organism vaccine candidate using the established sporozoite challenge model. GAPs might provide a safe and reproducible platform to develop an efficacious whole-cell malaria vaccine that prevents infection at the preerythrocytic stage.

BACKGROUND A subset of the Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1(SM)) is involved in the cytoadherence of P. falciparum-infected red blood cells (iRBC) contributing to the pathogenesis of severe disease among young children in malaria endemic areas. The PfEMP1(SM) are encoded by group A var genes that are composed of a more constrained range of amino acid sequences than groups B and C var genes encoding PfEMP1(UM) associated with uncomplicated malaria. Also, unlike var genes from groups B and C, those from group A do not have sequences consistent with CD36 binding--a major cytoadhesion phenotype of P. falciparum isolates. METHODS A 3D7 PfEMP1(SM) sub-line (3D7(SM)) expressing VAR4 (PFD1235w/MAL8P1.207) was selected for binding to CD36. The protein expression of this parasite line was monitored by surface staining of iRBC using VAR4-specific antibodies. The serological phenotype of the 3D7(SM) parasites was determined by flow cytometry using malaria semi-immune and immune plasma and transcription of the 59 var genes in 3D7 were analysed by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) using var-specific primers. RESULTS A selection-induced increased adhesion of 3D7(SM) iRBC to CD36 resulted in a reduced var4 transcription and VAR4 surface expression. CONCLUSION VAR4 is not involved in CD36 adhesion. The current findings are consistent with the notion that CD36 adhesion is not associated with particular virulent parasite phenotypes, such as those believed to be exhibited by VAR4 expressing parasites.

In the Saimiri sciureus monkey, erythrocytes infected with the varO antigenic variant of the Plasmodium falciparum Palo Alto 89F5 clone bind uninfected red blood cells ("rosetting"), form autoagglutinates and display a high multiplication rate, three phenotypic characteristics associated with severe malaria in human patients. We report here that varO parasites express a var gene presenting the characteristics of group A var genes, and show that the varO-DBL1alpha1 domain is implicated in the rosetting of both Saimiri sciureus and human erythrocytes. The soluble varO-NTS-DBL1alpha1 recombinant domain, produced in the baculovirus/insect cell system, induced high titers of antibodies that reacted with varO-infected red blood cells and disrupted varO rosettes. VarO parasites were culture-adapted in vitro using human erythrocytes. They formed rosettes and autoagglutinates, displayed the same surface serotype and expressed the same varO gene as the monkey-propagated parasites. To develop an in vitro model with highly homogeneous varO parasites, rosette purification was combined with positive selection by panning on a varO-NTS-DBL1alpha1-specific mouse monoclonal antibody. These single variant, clonal parasites were used to analyse seroprevalence to varO at the village level in a holoendemic setting (Dielmo, Senegal). We found 93.6%(95% CI: 89.7-96.4) seroprevalence of varO surface-reacting antibodies and 86.7%(95%CI: 82.8-91.6) seroprevalence to the recombinant NTS-DBL1alpha1 domain, with virtually all permanent residents having seroconverted by the age of five years. These data imply that varO represents a relevant in vivo/in vitro model of rosetting and autoagglutination for the rationale development of vaccine candidates and therapeutic strategies aimed at preventing malaria pathology.

Among the few vaccine candidates under development, apical membrane antigen (AMA-1) of Plasmodium falciparum is one of the most promising erythrocyte stage malaria vaccine candidates under consideration. The overall structure of AMA-1 appears to be conserved as compared to other surface proteins, but there are numerous amino acid substitutions identified among different P. falciparum isolates. Antisera raised against recombinant AMA-1 or naturally acquired human antibodies were strongly inhibitory only towards homologous parasites. In an attempt to examine the strain specificity of antibodies elicited to AMA-1, we have cloned, expressed and purified two allelic variants of domain I+II of AMA-1 ectodomain from Indian P. falciparum isolates in bacteria. One of these is a new haplotype not reported so far and varies in 18 aa positions from the geographically diverse forms 3D7 and 15 from FVO. Refolded proteins were recognized by a conformation specific monoclonal antibody 4G2.dc1 and hyper immune sera. Immunization of mice and rabbits with the purified proteins using CFA/IFA adjuvant generated high titer polyclonal antibodies. Both the alleles induced high levels of IgG1, IgG2a and IgG2b and a low level of IgG3 in mice. Lymphocyte proliferation assays using splenocytes from immunized mice showed significant proliferative responses and cytokines interleukin-2 (IL-2), IL-4, IL-10 and IFN-gamma presence in the culture supernatants. The anti-AMA-1 rabbit antibodies obtained with both the proteins were active in an in vitro parasite growth invasion/inhibition assay. These results suggest that recombinant AMA-1 domain I+II formulated with CFA/IFA adjuvant elicited cellular and humoral responses and is capable of inducing high titer invasion inhibitory antibodies supporting further development of this vaccine candidate.