J. Alexandra Rowe

Blood group O protects against severe Plasmodium falciparum malaria through the mechanism of reduced rosetting

Malaria has been a major selective force on the human population, and several erythrocyte polymorphisms have evolved that confer resistance to severe malaria. Plasmodium falciparum rosetting, a parasite virulence phenotype associated with severe malaria, is reduced in blood group O erythrocytes compared with groups A, B, and AB, but the contribution of the ABO blood group system to protection against severe malaria has received little attention. We hypothesized that blood group O may confer resistance to severe falciparum malaria through the mechanism of reduced rosetting. In a matched case-control study of 567 Malian children, we found that group O was present in only 21% of severe malaria cases compared with 44–45% of uncomplicated malaria controls and healthy controls. Group O was associated with a 66% reduction in the odds of developing severe malaria compared with the non-O blood groups (odds ratio 0.34, 95% confidence interval 0.19–0.61, P < 0.0005, severe cases versus uncomplicated malaria controls). In the same sample set, P. falciparum rosetting was reduced in parasite isolates from group O children compared with isolates from the non-O blood groups (P = 0.003, Kruskal–Wallis test). Statistical analysis indicated a significant interaction between host ABO blood group and parasite rosette frequency that supports the hypothesis that the protective effect of group O operates through the mechanism of reduced P. falciparum rosetting. This work provides insights into malaria pathogenesis and suggests that the selective pressure imposed by malaria may contribute to the variable global distribution of ABO blood groups in the human population.

Adhesion of Plasmodium falciparum-infected erythrocytes to human cells: molecular mechanisms and therapeutic implications.

Severe malaria has a high mortality rate (15–20%) despite treatment with effective antimalarial drugs. Adjunctive therapies for severe malaria that target the underlying disease process are therefore urgently required. Adhesion of erythrocytes infected with Plasmodium falciparum to human cells has a key role in the pathogenesis of life-threatening malaria and could be targeted with antiadhesion therapy. Parasite adhesion interactions include binding to endothelial cells (cytoadherence), rosetting with uninfected erythrocytes and platelet-mediated clumping of infected erythrocytes. Recent research has started to define the molecular mechanisms of parasite adhesion, and antiadhesion therapies are being explored. However, many fundamental questions regarding the role of parasite adhesion in severe malaria remain unanswered. There is strong evidence that rosetting contributes to severe malaria in sub-Saharan Africa; however, the identity of other parasite adhesion phenotypes that are implicated in disease pathogenesis remains unclear. In addition, the possibility of geographic variation in adhesion phenotypes causing severe malaria, linked to differences in malaria transmission levels and host immunity, has been neglected. Further research is needed to realise the untapped potential of antiadhesion adjunctive therapies, which could revolutionise the treatment of severe malaria and reduce the high mortality rate of the disease.

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.

Differential var gene transcription in Plasmodium falciparum isolates from patients with cerebral malaria compared to hyperparasitaemia

The Plasmodium falciparum variant erythrocyte surface antigens known as PfEMP1, encoded by the var gene family, are thought to play a crucial role in malaria pathogenesis because they mediate adhesion to host cells and immuno-modulation. Var genes have been divided into three major groups (A, B and C) and two intermediate groups (B/A and B/C) on the basis of their genomic location and upstream sequence. We analysed expressed sequence tags of the var gene DBLα domain to investigate var gene transcription in relation to disease severity in Malian children. We found that P. falciparum isolates from children with cerebral malaria (unrousable coma) predominantly transcribe var genes with DBLα1-like domains that are characteristic of Group A or B/A var genes. In contrast, isolates from children with equally high parasite burdens but no symptoms or signs of severe malaria (hyperparasitaemia patients) predominantly transcribe var genes with DBLα0-like domains that are characteristic of the B and C-related var gene groups. These results suggest that var genes with DBLα1-like domains (Group A or B/A) may be implicated in the pathogenesis of cerebral malaria, while var genes with DBLα0-like domains promote less virulent malaria infections.

Virulence of malaria is associated with differential expression of Plasmodium falciparum var gene subgroups in a case-control study

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a major pathogenicity factor in falciparum malaria that mediates cytoadherence. PfEMP1 is encoded by approximately 60 var genes per haploid genome. Most var genes are grouped into 3 subgroups: A, B, and C. Evidence is emerging that the specific expression of these subgroups has clinical significance. Using field samples from children from Papua New Guinea with severe, mild, and asymptomatic malaria, we compared proportions of transcripts of var groups, as determined by quantitative polymerase chain reaction. We found a significantly higher proportion of var group B transcripts in children with clinical malaria (mild and severe), whereas a large proportion of var group C transcripts was found in asymptomatic children. These data from naturally infected children clearly show that major differences exist in var gene expression between parasites causing clinical disease and those causing asymptomatic infections. Furthermore, parasites forming rosettes showed a significant up-regulation of var group A transcripts.

A subset of group A-like var genes encodes the malaria parasite ligands for binding to human brain endothelial cells.

Cerebral malaria is the most deadly manifestation of infection with Plasmodium falciparum. The pathology of cerebral malaria is characterized by the accumulation of infected erythrocytes (IEs) in the microvasculature of the brain caused by parasite adhesins on the surface of IEs binding to human receptors on microvascular endothelial cells. The parasite and host molecules involved in this interaction are unknown. We selected three P. falciparum strains (HB3, 3D7, and IT/FCR3) for binding to a human brain endothelial cell line (HBEC-5i). The whole transcriptome of isogenic pairs of selected and unselected parasites was analyzed using a variant surface antigen-supplemented microarray chip. After selection, the most highly and consistently up-regulated genes were a subset of group A-like var genes (HB3var3, 3D7_PFD0020c, ITvar7, and ITvar19) that showed 11- to >100-fold increased transcription levels. These var genes encode P. falciparum erythrocyte membrane protein (PfEMP)1 variants with distinct N-terminal domain types (domain cassette 8 or domain cassette 13). Antibodies to HB3var3 and PFD0020c recognized the surface of live IEs and blocked binding to HBEC-5i, thereby confirming the adhesive function of these variants. The clinical in vivo relevance of the HBEC-selected parasites was supported by significantly higher surface recognition of HBEC-selected parasites compared with unselected parasites by antibodies from young African children suffering cerebral malaria (Mann–Whitney test, P = 0.029) but not by antibodies from controls with uncomplicated malaria (Mann–Whitney test, P = 0.58). This work describes a binding phenotype for virulence-associated group A P. falciparum erythrocyte membrane protein 1 variants and identifies targets for interventions to treat or prevent cerebral malaria.

A well‐conserved Plasmodium falciparum var gene shows an unusual stage‐specific transcript pattern

The var multicopy gene family encodes Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) variant antigens, which, through their ability to adhere to a variety of host receptors, are thought to be important virulence factors. The predominant expression of a single cytoadherent PfEMP1 type on an infected red blood cell, and the switching between different PfEMP1 types to evade host protective antibody responses, are processes thought to be controlled at the transcriptional level. Contradictory data have been published on the timing of var gene transcription. Reverse transcription‐polymerase chain reaction (RT‐PCR) data suggested that transcription of the predominant var gene occurs in the later (pigmented trophozoite) stages, whereas Northern blot data indicated such transcripts only in early (ring) stages. We investigated this discrepancy by Northern blot, with probes covering a diverse var gene repertoire. We confirm that almost all var transcript types were detected only in ring stages. However, one type, the well‐conserved varCSA transcript, was present constitutively in different laboratory parasites and does not appear to undergo antigenic variation. Although varCSA has been shown to encode a chondroitin sulphate A (CSA)‐binding PfEMP1, we find that the presence of full‐length varCSA transcripts does not correlate with the CSA‐binding phenotype.

Mapping of the Region of Complement Receptor (CR) 1 Required for Plasmodium falciparum Rosetting and Demonstration of the Importance of CR1 in Rosetting in Field Isolates

The malaria parasite Plasmodium falciparum induces a number of novel adhesion properties in the erythrocytes that it infects. One of these properties, the ability of infected erythrocytes to bind uninfected erythrocytes to form rosettes, is associated with severe malaria and may play a direct role in the pathogenesis of disease. Previous work has shown that erythrocytes deficient in complement receptor (CR) 1 (CR1, CD35; C3b/C4b receptor) have greatly reduced rosetting capacity, indicating an essential role for CR1 in rosette formation. Using deletion mutants and mAbs, we have localized the region of CR1 required for the formation of P. falciparum rosettes to the area of long homologous repeat regions B and C that also acts as the binding site for the activated complement component C3b. This result raises the possibility that C3b could be an intermediary in rosetting, bridging between the infected erythrocyte and CR1. We were able to exclude this hypothesis, however, as parasites grown in C3-deficient human serum formed rosettes normally. We have also shown in this report that rosettes can be reversed by mAb J3B11 that recognizes the C3b binding site of CR1. This rosette-reversing activity was demonstrated in a range of laboratory-adapted parasite strains and field isolates from Kenya and Malawi. Thus, we have mapped the region of CR1 required for rosetting and demonstrated that the CR1-dependent rosetting mechanism occurs commonly in P. falciparum isolates, and could therefore be a potential target for future therapeutic interventions to treat severe malaria.

Identification of a Conserved Plasmodium falciparum var Gene Implicated in Malaria in Pregnancy

The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family is a highly polymorphic class of variant surface antigens encoded by var genes that play an important role in malaria pathogenesis. This report describes the unexpected finding that 1 of the var genes encoding a PfEMP1 variant that binds to the host receptor chondroitin sulfate A (CSA) and is implicated in malaria in pregnancy is well conserved among P. falciparum isolates worldwide. The N-terminal domains of this PfEMP1 variant are especially highly conserved, whereas the functional CSA binding domain is more variable. Analysis of var gene expression in placental parasites from primigravid women in Malawi did not support a role for this conserved gene in placental infection but identified a second commonly occurring var gene. These results indicate the need for reevaluation of previous assumptions of a minimal overlap between var gene repertoires from different parasite isolates.

Transcribed var Genes Associated with Placental Malaria in Malawian Women

ABSTRACT Determining the diversity of PfEMP1 sequences expressed by Plasmodium falciparum-infected erythrocytes isolated from placentas is important for attempts to develop a pregnancy-specific malaria vaccine. The DBLγ and var2csa DBL3x domains of PfEMP1 molecules are believed to mediate placental sequestration of infected erythrocytes, so the sequences encoding these domains were amplified from the cDNAs of placental parasites by using degenerate oligonucleotides. The levels of specific var cDNAs were then determined by quantitative reverse transcription-PCR. Homologues of var2csa DBL3x were the predominant sequences amplified from the cDNAs of most placental but not most childrens parasites. There was 56% identity between all placental var2csa sequences. Many different DBLγ domains were amplified from the cDNAs of placental and childrens isolates. var2csa transcripts were the most abundant var transcripts of those tested in 11 of 12 placental isolates and 1 of 6 childrens isolates. Gravidity did not affect the levels of var2csa transcripts. We concluded that placental malaria is frequently associated with transcription of var2csa but that other var genes are also expressed, and parasites expressing high levels of var2csa are not restricted to pregnant women. The diversity of var2csa sequences may be important for understanding immunity and for the development of vaccines for malaria during pregnancy.