John C. Reeder

Targeted Gene Disruption Shows That Knobs Enable Malaria-Infected Red Cells to Cytoadhere under Physiological Shear Stress

Knobs at the surface of erythrocytes infected with Plasmodium falciparum have been proposed to be important in adherence of these cells to the vascular endothelium. This structure contains the knob-associated histidine-rich protein (KAHRP) and the adhesion receptor P. falciparum erythrocyte membrane protein 1. We have disrupted the gene encoding KAHRP and show that it is essential for knob formation. Knob-transfectants adhere to CD36 in static assays; when tested under flow conditions that mimic those of postcapillary venules, however, the binding to CD36 was dramatically reduced. These data suggest that knobs on P. falciparum-infected erythrocytes exert an important influence on adherence of parasitized-erythrocytes to microvascular endothelium, an important process in the pathogenesis of P. falciparum infections.

Plasmodium Vivax and Mixed Infections Are Associated with Severe Malaria in Children: A Prospective Cohort Study from Papua New Guinea

Background Severe malaria (SM) is classically associated with Plasmodium falciparum infection. Little information is available on the contribution of P. vivax to severe disease. There are some epidemiological indications that P. vivax or mixed infections protect against complications and deaths. A large morbidity surveillance conducted in an area where the four species coexist allowed us to estimate rates of SM among patients infected with one or several species. Methods and Findings This was a prospective cohort study conducted within the framework of the Malaria Vaccine Epidemiology and Evaluation Project. All presumptive malaria cases presenting at two rural health facilities over an 8-y period were investigated with history taking, clinical examination, and laboratory assessment. Case definition of SM was based on the World Health Organization (WHO) criteria adapted for the setting (i.e., clinical diagnosis of malaria associated with asexual blood stage parasitaemia and recent history of fits, or coma, or respiratory distress, or anaemia [haemoglobin < 5 g/dl]). Out of 17,201 presumptive malaria cases, 9,537 (55%) had a confirmed Plasmodium parasitaemia. Among those, 6.2% (95% confidence interval [CI] 5.7%–6.8%) fulfilled the case definition of SM, most of them in children <5 y. In this age group, the proportion of SM was 11.7% (10.4%–13.2%) for P. falciparum, 8.8% (7.1%–10.7%) for P. vivax, and 17.3% (11.7%–24.2%) for mixed P. falciparum and P. vivax infections. P. vivax SM presented more often with respiratory distress than did P. falciparum (60% versus 41%, p = 0.002), but less often with anaemia (19% versus 41%, p = 0.0001). Conclusion P. vivax monoinfections as well as mixed Plasmodium infections are associated with SM. There is no indication that mixed infections protected against SM. Interventions targeted toward P. falciparum only might be insufficient to eliminate the overall malaria burden, and especially severe disease, in areas where P. falciparum and P. vivax coexist.

Adhesion of Plasmodium falciparum-infected erythrocytes to hyaluronic acid in placental malaria

Infection with Plasmodium falciparum during pregnancy leads to the accumulation of parasite-infected erythrocytes in the placenta, and is associated with excess perinatal mortality, premature delivery and intrauterine growth retardation in the infant, as well as increased maternal mortality and morbidity. P. falciparum can adhere to specific receptors on host cells, an important virulence factor enabling parasites to accumulate in various organs. We report here that most P. falciparum isolates from infected placentae can bind to hyaluronic acid, a newly discovered receptor for parasite adhesion that is present on the placental lining. In laboratory isolates selected for specific high-level adhesion, binding to hyaluronic acid could be inhibited by dodecamer or larger oligosaccharide fragments or polysaccharides, treatment of immobilized receptor with hyaluronidase, or treatment of infected erythrocytes with trypsin. In vitro flow-based assays demonstrated that high levels of adhesion occurred at low wall shear stress, conditions thought to prevail in the placenta. Our findings indicate that adhesion to hyaluronic acid is involved in mediating placental parasite accumulation, thus changing the present understanding of the mechanisms of placental infection, with implications for the development of therapeutic and preventative interventions.

Genome-wide and fine-resolution association analysis of malaria in West Africa.

We report a genome-wide association (GWA) study of severe malaria in The Gambia. The initial GWA scan included 2,500 children genotyped on the Affymetrix 500K GeneChip, and a replication study included 3,400 children. We used this to examine the performance of GWA methods in Africa. We found considerable population stratification, and also that signals of association at known malaria resistance loci were greatly attenuated owing to weak linkage disequilibrium (LD). To investigate possible solutions to the problem of low LD, we focused on the HbS locus, sequencing this region of the genome in 62 Gambian individuals and then using these data to conduct multipoint imputation in the GWA samples. This increased the signal of association, from P = 4 × 10−7 to P = 4 × 10−14, with the peak of the signal located precisely at the HbS causal variant. Our findings provide proof of principle that fine-resolution multipoint imputation, based on population-specific sequencing data, can substantially boost authentic GWA signals and enable fine mapping of causal variants in African populations.

Plasmodium falciparum erythrocyte invasion through glycophorin C and selection for Gerbich negativity in human populations

Geographic overlap between malaria and the occurrence of mutant hemoglobin and erythrocyte surface proteins has indicated that polymorphisms in human genes have been selected by severe malaria. Deletion of exon 3 in the glycophorin C gene (called GYPCΔex3 here) has been found in Melanesians; this alteration changes the serologic phenotype of the Gerbich (Ge) blood group system, resulting in Ge negativity. The GYPCΔex3 allele reaches a high frequency (46.5%) in coastal areas of Papua New Guinea where malaria is hyperendemic. The Plasmodium falciparum erythrocyte-binding antigen 140 (EBA140, also known as BAEBL) binds with high affinity to the surface of human erythrocytes. Here we show that the receptor for EBA140 is glycophorin C (GYPC) and that this interaction mediates a principal P. falciparum invasion pathway into human erythrocytes. EBA140 does not bind to GYPC in Ge-negative erythrocytes, nor can P. falciparum invade such cells using this invasion pathway. This provides compelling evidence that Ge negativity has arisen in Melanesian populations through natural selection by severe malaria.

A var gene promoter controls allelic exclusion of virulence genes in Plasmodium falciparum malaria

Mono-allelic expression of gene families is used by many organisms to mediate phenotypic variation of surface proteins. In the apicomplexan parasite Plasmodium falciparum, responsible for the severe form of malaria in humans, this is exemplified by antigenic variation of the highly polymorphic P. falciparum erythrocyte membrane protein 1 (PfEMP1). PfEMP1, encoded by the 60-member var gene family, represents a major virulence factor due to its central role in immune evasion and intravascular parasite sequestration. Mutually exclusive expression of PfEMP1 is controlled by epigenetic mechanisms involving chromatin modification and perinuclear var locus repositioning. Here we show that a var promoter mediates the nucleation and spreading of stably inherited silenced chromatin. Transcriptional activation of this promoter occurs at the nuclear periphery in association with chromosome-end clusters. Additionally, the var promoter sequence is sufficient to infiltrate a transgene into the allelic exclusion programme of var gene expression, as transcriptional activation of this transgene results in silencing of endogenous var gene transcription. These results show that a var promoter is sufficient for epigenetic silencing and mono-allelic transcription of this virulence gene family, and are fundamental for our understanding of antigenic variation in P. falciparum. Furthermore, the PfEMP1 knockdown parasites obtained in this study will be important tools to increase our understanding of P. falciparum-mediated virulence and immune evasion.

A human complement receptor 1 polymorphism that reduces Plasmodium falciparum rosetting confers protection against severe malaria

Parasitized red blood cells (RBCs) from children suffering from severe malaria often adhere to complement receptor 1 (CR1) on uninfected RBCs to form clumps of cells known as “rosettes.” Despite a well documented association between rosetting and severe malaria, it is controversial whether rosetting is a cause or a correlate of parasite virulence. CR1-deficient RBC show greatly reduced rosetting; therefore, we hypothesized that, if rosetting is a direct cause of malaria pathology, CR1-deficient individuals should be protected against severe disease. In this study, we show that RBC CR1 deficiency occurs in up to 80% of healthy individuals from the malaria-endemic regions of Papua New Guinea. This RBC CR1 deficiency is associated with polymorphisms in the CR1 gene and, unexpectedly, with α-thalassemia, a common genetic disorder in Melanesian populations. Analysis of a case-control study demonstrated that the CR1 polymorphisms and α-thalassemia independently confer protection against severe malaria. We have therefore identified CR1 as a new malaria resistance gene and provided compelling evidence that rosetting is an important parasite virulence phenotype that should be a target for drug and vaccine development.

Targeted mutagenesis of Plasmodium falciparum erythrocyte membrane protein 3 (PfEMP3) disrupts cytoadherence of malaria‐infected red blood cells

Adhesion of parasite‐infected red blood cells to the vascular endothelium is a critical event in the pathogenesis of malaria caused by Plasmodium falciparum. Adherence is mediated by the variant erythrocyte membrane protein 1 (PfEMP1). Another protein, erythrocyte membrane protein‐3 (PfEMP3), is deposited under the membrane of the parasite‐infected erythrocyte but its function is unknown. Here we show that mutation of PfEMP3 disrupts transfer of PfEMP1 to the outside of the P.falciparum‐infected cell. Truncation of the C‐terminal end of PfEMP3 by transfection prevents distribution of this large (>300 kDa) protein around the membrane but does not disrupt trafficking of the protein from the parasite to the cytoplasmic face of the erythrocyte membrane. The truncated PfEMP3 accumulates in structures that appear to be associated with the erythrocyte membrane. We show that accumulation of mutated PfEMP3 blocks the transfer of PfEMP1 onto the outside of the parasitized cell surface and suggest that these proteins traffic through an erythrocyte membrane‐associated compartment that is involved in the transfer of PfEMP1 to the surface of the parasite‐infected red blood cell.

Reduced risk of clinical malaria in children infected with multiple clones of Plasmodium falciparum in a highly endemic area: a prospective community study.

A prospective community study in a highly malaria endemic area of Papua New Guinea found that infection with multiple Plasmodium falciparum genotypes was an indicator of lowered risk of subsequent clinical attack. The results suggest that concurrent or very recent infections provide protection from superinfecting parasites. The finding of an association between reduced risk of clinical malaria and infection with parasites of merozoite surface protein 1 (MSP-1) type RO33 or MSP-2 type 3D7 further suggests that the concomitant immunity is, at least in part, a consequence of a response to these major merozoite surface proteins.

Geographical Structure of Diversity and Differences between Symptomatic and Asymptomatic Infections for Plasmodium falciparum Vaccine Candidate AMA1

ABSTRACT Plasmodium falciparum apical membrane antigen 1 (AMA1) is a prime malaria vaccine candidate. Antigenic diversity within parasite populations is one of the main factors potentially limiting the efficacy of any asexual-stage vaccine, including one based on AMA1. The DNA coding for the most variable region of this antigen, domain I, was sequenced in 168 samples from the Wosera region of Papua New Guinea, including samples from symptomatic and asymptomatic infections. Neutrality tests applied to these sequences provided strong evidence of selective pressure operating on the sequence of ama1 domain I, consistent with AMA1 being a target of protective immunity. Similarly, a peculiar pattern of geographical diversity and the particular substitutions found were suggestive of strong constraints acting on the evolution of AMA1 at the population level, probably as a result of immune pressure. In addition, a strong imbalance between symptomatic and asymptomatic infections was detected in the frequency of particular residues at certain polymorphic positions, pointing to AMA1 as being one of the determinants of the morbidity associated with a particular strain. The information yielded by this study has implications for the design and assessment of AMA1-based vaccines and provides additional data supporting the importance of AMA1 as a malaria vaccine candidate.