PfEMP1 - A Parasite Protein Family of Key Importance in Plasmodium falciparum Malaria Immunity and Pathogenesis.

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PfEMP1 e A Parasite Protein Family of Key Importance in Plasmodium falciparum Malaria Immunity and Pathogenesis Lars Hviid1 and Anja TR. Jensen Centre for Medical Parasitology, University of Copenhagen and Copenhagen University Hospital (Rigshospitalet), Copenhagen, Denmark 1 Corresponding author: E-mail: [email protected]

Contents 1. Introduction 2. The Structure of var Genes and PfEMP1 Proteins 2.1 Primary structure 2.2 Secondary structure 2.3 Tertiary structure 2.4 Quaternary structure 3. The Function of PfEMP1 3.1 Type 3 PfEMP1 3.2 DC4-type PfEMP1 3.3 DC5-type PfEMP1 3.4 DC8- and DC13-type PfEMP1 3.5 VAR2CSA-type PfEMP1 3.6 Non-DC4-mediated adhesion of PfEMP1 to ICAM-1 3.7 PfEMP1-mediated adhesion of IEs to CD36 3.8 PfEMP1 and rosetting 3.9 PfEMP1 and gametocytes 4. Structural Organization of PfEMP1 at the IE Surface 5. Regulation of var Gene Transcription and Switching 5.1 Transcription of var genes 5.2 Switching transcription from one var gene to another 6. PfEMP1-Mediated Pathogenesis 6.1 Cerebral malaria 6.2 Placental malaria 7. PfEMP1-Specific Immune Responses 8. PfEMP1 and Vaccination against Malaria 9. Conclusions and Future Directions Acknowledgements References Advances in Parasitology, Volume 88 ISSN: 0065-308X http://dx.doi.org/10.1016/bs.apar.2015.02.004

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Lars Hviid and Anja TR. Jensen

Abstract Plasmodium falciparum causes the most severe form of malaria and is responsible for essentially all malaria-related deaths. The accumulation in various tissues of erythrocytes infected by mature P. falciparum parasites can lead to circulatory disturbances and inflammation, and is thought to be a central element in the pathogenesis of the disease. It is mediated by the interaction of parasite ligands on the erythrocyte surface and a range of host receptor molecules in many organs and tissues. Among several proteins and protein families implicated in this process, the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family of high-molecular weight and highly variable antigens appears to be the most prominent. In this chapter, we aim to provide a systematic overview of the current knowledge about these proteins, their structure, their function, how they are presented on the erythrocyte surface, and how the var genes encoding them are regulated. The role of PfEMP1 in the pathogenesis of malaria, PfEMP1-specific immune responses, and the prospect of PfEMP1-specific vaccination against malaria are also covered briefly.

Abbreviations CIDR CSA DBL IE MS PfEMP1

Cysteine-rich interdomain region Chondroitin sulphate 1 Duffy-binding-like Infected erythrocyte Mass spectrometry Plasmodium falciparum erythrocyte membrane protein 1

1. INTRODUCTION Several species of malaria parasites cause the erythrocytes they infect to become sticky (reviewed by Berendt et al., 1990). In Plasmodium falciparum, this process is so efficient that only erythrocytes infected with young parasites (the so-called ring-stages) are present in the peripheral circulation of infected individuals1, a fact that has been known for well over 100 years (Marchiafava and Bignami, 1894). The reason is that all P. falciparuminfected erythrocytes (IEs) older than approximately 18 h sequester in the post-capillary venules of various tissues and organs for the remainder of the parasite’s 48-h asexual multiplication cycle (MacPherson et al., 1985; Miller, 1969). This key pathophysiological event can have dire 1

Mature gametocytes constitute an exception to this rule as discussed below.

ARTICLE IN PRESS The PfEMP1 Antigen Family

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consequences when it occurs in organs such as the brain or the placenta (Bignami and Bastianelli, 1889; Clark, 1915). By the late 1980s it was clear that the tissue-specific sequestration of P. falciparum-IEs involves highmolecular weight (>200 kD) and strain-specific parasite molecules displayed on electron-dense IE surface protrusions (‘knobs’) (Langreth and Reese, 1979; Leech et al., 1984b; Udeinya et al., 1983). One such trypsin-sensitive and high-molecular weight antigen was named P. falciparum erythrocyte membrane protein 1 (PfEMP1) by Howard et al. (1988). Less than a decade later, three independent studies using different experimental approaches and published back-to-back demonstrated that PfEMP1 was in fact a family of proteins encoded by the var multigene family (Baruch et al., 1995; Smith et al., 1995; Su et al., 1995). This review is an attempt to summarize what is known about these proteins and in particular the relation between their structure and their function. Although several other multigene families have also been identified, their role in IE adhesion is more contentious. Readers seeking overviews about those families and the proteins they encode, or on serologically defined variant surface antigens, are referred to separate reviews in the literature (e.g. Chan et al., 2014; Hviid, 2005; Jemmely et al., 2010).

2. THE STRUCTURE OF VAR GENES AND PfEMP1 PROTEINS PfEMP1 is a family of high-molecular weight proteins (approximately 200e450 kD) anchored in the surface membrane of P. falciparum-IEs. They are encoded by the w60 two-exon var genes in the haploid P. falciparum genome (Gardner et al., 2002). Exon 1 encodes the extracellular part of the proteins, while the short transmembrane domain and the intracellular terminal segment are encoded by exon 2 (Figure 1).

2.1 Primary structure In contrast to the relatively conserved exon 2, there is very extensive nucleotide diversity in exon 1 of the var genes, and the primary structure (the amino acid sequence) of the extracellular part of PfEMP1 proteins is therefore also highly variable. This applies both within single genomes (intraclonal variation) and between genomes (interclonal diversity). Despite the variability, the var genes (and the PfEMP1 proteins they encode) are often divided into groups according to the chromosomal location, upstream promoter sequence (ups), and direction of transcription of the var genes. Group

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Figure 1 Chromosomal organization of Plasmodium falciparum var genes and P. falciparum erythrocyte membrane protein 1 (PfEMP1) domain architecture. (a) Group A and B var genes are located in subtelomeric regions of all chromosomes, but are transcribed in opposite directions. Group C var genes are found in central chromosomal regions. (b) PfEMP1 proteins are built of different subtypes of Duffy-bindinglike (DBL) and cysteine-rich interdomain region (CIDR) domains. Groups B and Group C PfEMP1 proteins predominantly have a four-domain structure, whereas larger (mainly Group A) PfEMP1 proteins have additional DBL domains following the first or second DBL-CIDR domain pair. (c) Plasmodium falciparum genomes encode tandem domain arrangements (domain cassettes; DC). These DCs are linked to different known adhesion phenotypes as indicated. DC8 is a chimeric gene between a group A and group B var gene. TM, thrombomodulin; ATS, acidic, terminal sequence; UPS, upstream promoter sequence. Figure modified from Smith et al. (2013).

A (10 genes in P. falciparum 3D7), Group B (22 genes), and Group B/A var genes (4 genes) are found in the subtelomeric regions of all chromosomes, but are transcribed in opposite directions (Group A genes towards the telomere, Group B and Group B/A var genes towards the centromere) (Figure 1). In contrast, Group C (13 genes in P. falciparum 3D7) and Group B/C var genes (9 genes) are typically found in internal regions of chromosome 4, 7, 8 and 12 (Gardner et al., 2002; Kraemer and Smith, 2003; Lavstsen et al., 2003; Rask et al., 2010).


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2.2 Secondary structure The secondary structure of the PfEMP1 proteins is modular, as the amino acid sequence can be divided into 2e10 Duffy-binding-like (DBL) domains and cysteine-rich interdomain regions (CIDR). The DBL domains are homologous to adhesive domains in P. falciparum EBA-175 and the Duffy-binding proteins of Plasmodium vivax and Plasmodium knowlesi (Peterson et al., 1995), while CIDR domains are characterized by conserved cysteine-rich motifs (Baruch et al., 1997). DBL domains can be divided into three structural subdomains (Figure 2) with either a mixed helix-sheet structure (S1) or composed of helix bundles (S2 and S3) (Smith et al., 2000). Disulphide bonds between conserved cysteine residues in the subdomains hold them together (Higgins, 2008b). Extensive recombination events at a recombination hot spot between S2 and S3 appear to have resulted in the formation of a number of homology blocks (HBs) and ultimately shaping of the PfEMP1 repertoire (Rask et al., 2010). Similar HBs can be found in CIDR domains, and DBL and CIDR domains can be divided into seven (a, b, g, d, ε, x, x) and three (a, b, g) main sequence classes, respectively, based on the HB arrangement. Each of these sequence classes can be further subdivided (Rask et al., 2010).

Figure 2 Modelled structure of PFD1235w Duffy-binding-like b (DBLb). (a) The DBL structure consists of subdomain 1 (S1, orange (light gray in print versions)) with mixed helix-sheet structure and two helix bundles subdomain 2 (S2; magenta (gray in print versions)) and subdomain 3 (S3; green (light gray in print versions)) (Higgins, 2008b; Singh et al., 2006). The ICAM-1-binding site of PFD1235w is located in S3 (Bengtsson et al., 2013). (b) Side chains shown for conserved residues as defined by Rask et al. (2010). (c) Schematic showing position of the five homology blocks (HB) in DBL domains. (Redrawn from Rask et al. (2010).) (d) The same HBs as in (c) shown on the modelled structure of PFD1235w-DBLb_D4.

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All PfEMP1 subfamilies except type 3 (see Section 3.1) and VAR2CSA (see Section 3.5) have a head structure at their N-terminus composed of semi-conserved DBLa domain and a CIDR domain (Gardner et al., 2002). The head structure is followed by a second, more diverse, DBLCIDR pair in most PfEMP1 proteins belonging to Groups B, B/C, and C. Group A and B/A PfEMP1 proteins contain additional DBL domains upstream and/or downstream of the second DBL-CIDR pair, and are thus composed of a total of 7e10 extracellular domains (Figure 1). The combination of DBL and CIDR domains in any given PfEMP1 protein is not random, and certain domains tend to occur together (Figure 1), forming what is now often referred to as domain cassettes (DCs) (Rask et al., 2010; Trimnell et al., 2006). Some DCs are composed of only a few domains, while others span entire PfEMP1 proteins. Their presence often predicts the receptor specificity of the PfEMP1 concerned (Section 3).

2.3 Tertiary structure Crystal structure analysis of single CIDRa (Klein et al., 2008) and DBL domains (Gangnard et al., 2013; Higgins, 2008a; Juillerat et al., 2011; Khunrae et al., 2009; Singh et al., 2008) and a two-domain DBLa-CIDRg construct (Vigan-Womas et al., 2012) have shown PfEMP1 domains to be composed of bundles of a-helices connected by flexible and highly variable loops (Figure 2). These act as a scaffold on which sequence variation takes place.

2.4 Quaternary structure The overall shape of the entire ectodomain is only known for a few PfEMP1 proteins. In the absence of full-length PfEMP1 crystal structures, what is known is based on small-angle X-ray spectrometry analysis of recombinant full-length proteins in solutions. The available low-resolution data include the Group A protein HB3VAR06 (Stevenson et al., in press), the Group B protein IT4VAR13 (Brown et al., 2013), and two VAR2CSA-type PfEMP1 molecules (Clausen et al., 2012; Srivastava et al., 2010). HB3VAR06, which mediates rosetting (see Section 3.8) and binds nonimmune IgM (Ghumra et al., 2012; Stevenson et al., submitted for publication), and the Cluster of Differentiation 36 (CD36)-and intercellularcell adhesion molecule 1 (ICAM-1)-binding IT4VAR13 (Avril et al., 2012; Howell et al., 2008) can be seen as ‘classical’ PfEMP1 proteins. Both are elongated and zigzagshaped rigid molecules with a length of approximately 30 nm. In contrast, the VAR2CSA-type PfEMP1 that mediates adhesion to CSA in the placenta


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(Salanti et al., 2003, 2004) has a more compact and globular shape, with a length of about 20 nm. It is likely that the high-order structure of the proteins facilitate the interactions with their cognate receptors.

3. THE FUNCTION OF PfEMP1 Sequestration of mature IEs in the vasculature is of vital importance to P. falciparum, as it allows the erythrocytes deformed and stiffened by the developing parasites inside them to avoid being destroyed in the spleen (Cranston et al., 1984; Hommel et al., 1983). As the primary function of the PfEMP1 proteins is to mediate this IE sequestration (Baruch et al., 1996), PfEMP1 diversity is constrained by their role as ligands for receptor-mediated adhesion. This sets them apart from other parasite variant surface antigen families such as the variant surface glycoproteins of trypanosomes, whose primary function is simply to act as antigenic decoys frustrating the humoral immune system and delaying acquisition of protective antibodies (reviewed by Horn, 2014). The receptor specificity and other features of individual PfEMP1 proteins are closely related to their structural characteristics (Section 2). Relatively few endothelial surface molecules have been proposed to act as host receptors for adhesion of P. falciparum-IEs, despite the great diversity of the PfEMP1 proteins. It thus appears that many different PfEMP1 proteins have affinity for the same receptor, and recent studies indicate that receptor specificity is related to the presence of certain DCs (see Section 2.2). Some PfEMP1 clearly contain several adhesive domains with different receptor specificity (Chen et al., 2000; Janes et al., 2011), which would allow for adhesion of a given IE to several host receptors, either simultaneously or in succession (Esser et al., 2014). An example is the ability of some PfEMP1 to mediate adhesion to receptors on the surface of uninfected erythrocytes, which leads to the formation of rosettes (see Section 3.8), in addition to their affinity for endothelial receptors (Adams et al., 2014).

3.1 Type 3 PfEMP1 The type 3 subfamily (also known as VAR3) contains the smallest known PfEMP1, each composed of just two extracellular domains, an N-terminal hybrid DBLa1.3/DBLx domain and a C-terminal DBLε8 domain (Gardner et al., 2002). The DBLx-DBLε sequence, which is unique to this protein subfamily, is called DC3 (Rask et al., 2010).

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Most P. falciparum genomes examined contain several genes encoding DC3-type PfEMP1 (Trimnell et al., 2006), but despite this and the conserved nature of type 3 PfEMP1, their function and receptor specificity remain unknown. IE surface expression of type 3 PfEMP1 has been demonstrated (Wang et al., 2012), and specific antibody can be detected in a minority of P. falciparum-exposed individuals (Cham et al., 2009; Rottmann et al., 2006; Wang et al., 2012).

3.2 DC4-type PfEMP1 The DC4 is a cassette composed of three domains (DBLa1.1/1.4-CIDRa1.6DBLb3, Figure 1), and defines a subfamily of Group A PfEMP1 that mediates binding to ICAM-1 (CD54) but not to CD36 (Bengtsson et al., 2013). It was identified by a search for orthologs of the P. falciparum 3D7 var gene pfd1235w in parasites from Ghanaian malaria patients. That particular 3D7 gene was of interest, because an earlier study had shown that it was preferentially transcribed by P. falciparum 3D7-IEs selected for specific recognition by IgG in serum from semi-immune African children (Jensen et al., 2004). Furthermore, PFD1235w-specific IgG is acquired early in life by children living in areas with stable transmission of P. falciparum, and is associated with clinical protection from malaria (Jensen et al., 2004; Lusingu et al., 2006). Transcription of DC4-encoding var genes is higher in parasites from cerebral malaria patients than in parasites from patients without severe disease, indicating a role for DC4-mediated IE adhesion in the pathogenesis of cerebral malaria (Bengtsson et al., 2013; Lavstsen et al., 2012). This hypothesis is supported by the finding that in vitro selection for expression of DC4-containing PfEMP1 results in IEs that can adhere to ICAM-1, but not to CD36 (Bengtsson et al., 2013), in contrast to most other PfEMP1 with affinity for ICAM-1 (see Section 3.6). Furthermore, a particular motif in the C-terminal third of the DC4 DBLb3 domain e where the ICAM-1-binding site is located (Bengtsson et al., 2013) e is also present in other Group A PfEMP1 that do not contain DC4 (our unpublished data). Finally, the majority of these ICAM-1-binding PfEMP1 also contain CIDRa subtypes similar to the endothelial protein C receptor (EPCR)-binding CIDR subtypes (see Section 3.4).

3.3 DC5-type PfEMP1 This Group A subfamily is characterized by the presence of DC5 composed of DBLg12-DBLd5-CIDRb3/4-DBLb7/9 (Figure 1) (Rask et al., 2010).


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DC5-type PfEMP1, typified by PF11_0008 in P. falciparum 3D7, are more likely to be expressed in children with severe malaria than in children with uncomplicated malaria (Jensen et al., 2004). Antibodies to the DC5-type PfEMP1 are acquired early in life, and high levels of DC5-specific IgG are associated with normal haemoglobin levels and protection from malaria (Berger et al., 2013; Cham et al., 2009; Magistrado et al., 2007). It appears that IE adhesion to PECAM-1 (CD31), expressed by endothelial cells, monocytes, platelets and granulocytes (Baruch et al., 1995), is mediated by DC5 (Berger et al., 2013; Joergensen et al., 2010a).

3.4 DC8- and DC13-type PfEMP1 DC8 consists of four domains (DBLa2-CIDRa1.1-DBLb12-DBLg4/6, Figure 1) found among Group B PfEMP1, but appears to have evolved by recombination of ancestral Group A and Group B var genes (Lavstsen et al., 2012). DC13, which is found in some Group A PfEMP1 proteins, is composed of two domains (DBLa1.7 and CIDRa1.4). DC8- and DC13encoding var genes appear to be common, as they were found in six of seven fully sequenced P. falciparum genomes (Rask et al., 2010). The functional significance of these DCs became clear, when it was shown that P. falciparumIEs selected for adhesion to brain endothelial cells2 preferentially express PfEMP1 containing DC8 and DC13 (Avril et al., 2012; Claessens et al., 2012). Furthermore, parasites obtained from children with severe malaria (cerebral malaria and severe malarial anaemia) selectively transcribe DC8and DC13-encoding var genes (Lavstsen et al., 2012). DC8-specific antisera blocked adhesion of brain cell-adhering erythrocytes infected by parasites expressing DC8-type PfEMP1 (Claessens et al., 2012), and cerebral malaria convalescents were found to have particularly high levels of IgG specific for DC8-type PfEMP1þ IEs (Claessens et al., 2012). Finally, children living in malaria-endemic areas acquire DC8-specific IgG early in life (Avril et al., 2012; Lavstsen et al., 2012). The cognate receptor for DC8- and DC13-containing PfEMP1 has been identified as EPCR (Turner et al., 2013). PfEMP1 binding to EPCR is mediated by several CIDRa subtypes (CIDRa1.1, CIDRa1.4, CIDRa1.5 and CIDRa1.7) (Turner et al., 2013). EPCR is a transmembrane glycoprotein homologous to CD1/major histocompatibility complex molecules 2

In addition to their affinity for brain endothelial cells, DC8þ IEs also bind to endothelial cells from other tissues, such as lung, heart and dermis (Avril et al., 2012).

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(Fukudome and Esmon, 1994; Oganesyan et al., 2002). It is expressed on many cells types, including endothelium in many tissues (reviewed by Gleeson et al., 2012).

3.5 VAR2CSA-type PfEMP1 Members of the seven-domain VAR2CSA subfamily are composed of seven unusually structured domains that always occur together (Figure 1). The entire ectodomain thus constitutes a single DC. Three N-terminal DBLx (also known as DBLPAM) domains are followed by three DBLε domains, with a single CIDRPAM domain positioned between the second and third DBLx domain (Figure 1). VAR2CSA-type PfEMP1 thus lack the typical N-terminal head structure found in almost all other PfEMP1 proteins. The first VAR2CSA-type PfEMP1 (PFL0030c) was identified by analysis of changes in var gene transcription following selection of P. falciparum 3D7-IEs for adhesion to chondroitin sulphate A (CSA, also known as chondroitin-4-sulphate or C4S) (Salanti et al., 2003, 2004). CSA had been identified as an IE adhesion receptor almost a decade earlier (Rogerson et al., 1995), and shown to be of central importance in the pathogenesis of placental malaria (Fried and Duffy, 1996). One or more orthologs of the archetypical var2csa gene pfl0030c is present in all P. falciparum genomes studied so far (Rask et al., 2010; Salanti et al., 2003; Sander et al., 2009), as well as in the phylogenetically close chimpanzee parasite Plasmodium reichenowi (Trimnell et al., 2006). Several domains in VAR2CSA have affinity for CSA, but the full-length VAR2CSA binds several orders of magnitude better than any of its individual domain (Khunrae et al., 2010; Srivastava et al., 2010). This indicates that the receptor affinity of VAR2CSA-type PfEMP1 is critically dependent on the high-order conformation of the molecule (reviewed by Dahlb€ack et al., 2010). A minimal CSA-binding site, centred on the second DBLX domain, has similar affinity to the intact protein (Clausen et al., 2012; Dahlb€ack et al., 2011; Srivastava et al., 2011), although recent evidence suggests that cooperative adhesion mediated by several domains may also contribute to the high CSA-affinity of native VAR2CSA (Rieger et al., 2015). Although CSA is expressed at low density in many organs, VAR2CSA-expressing parasites are essentially confined to pregnant women. The tropism of CSAadhering IEs for the placenta is related to the particular sulphation pattern of CSA in the intervillous space (Achur et al., 2000; Alkhalil et al., 2000) and to the high density of CSA there (Rieger et al., 2015).


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While other ligands and receptors have been implicated in the accumulation of P. falciparum-IEs in the placenta, the vast bulk of the available evidence point to VAR2CSA-type PfEMP1 and CSA as the main ligande receptor pair involved (reviewed by Hviid, 2011).

3.6 Non-DC4-mediated adhesion of PfEMP1 to ICAM-1 ICAM-1 was originally identified as an IE adhesion receptor by Berendt et al. (1989), and IE adhesion to this receptor has repeatedly been implicated in the pathogenesis of cerebral malaria (see Section 6.1). However, until the discovery of DC4-type PfEMP1 (see Section 3.2), all but one ICAM-1-binding domain3 in PfEMP1 were diverse DBLb domains in Group B and Group C proteins (Howell et al., 2008; Janes et al., 2011; Oleinikov et al., 2009), and all of these appear to be under dual selection for binding to both ICAM-1 and CD36 (Cooke et al., 1994; Janes et al., 2011). Adhesion to CD36 is a common IE adhesion phenotype associated with uncomplicated malaria (Ochola et al., 2011). As IE adhesion to CD36 and ICAM-1 can act in synergy (Gray et al., 2003; Ho et al., 2000), it would appear that non-DC4 ICAM-1-binders evolved to adhere in tissues other than the brain (as CD36 is absent or sparse on cerebral endothelium (Turner et al., 1994; Wassmer et al., 2011)), in contrast to DC4-mediated adhesion to ICAM-1 (see Section 3.2). This could potentially resolve the equivocal evidence regarding the role of IE adhesion to ICAM-1 in the pathogenesis of cerebral malaria (see Section 6.1).

3.7 PfEMP1-mediated adhesion of IEs to CD36 The thrombospondin receptor CD36 was one of the earliest identified P. falciparum-IE adhesion receptors (Ockenhouse et al., 1989) with affinity for a high-molecular weight (approx. 270 kD) parasite ligand (Ockenhouse et al., 1991). We now know that binding to CD36 is a common IE adhesion phenotype and that it is mediated by CIDRa26 domains (Smith et al., 2013), which are widespread among PfEMP1 (Baruch et al., 1997; Gamain et al., 2001; Robinson et al., 2003) and that expression of CD36-adherent PfEMP1 is characteristic of P. falciparum parasites obtained from patients with uncomplicated malaria (Cabrera et al., 2014; Ochola et al., 2011). 3

The exception is a DBLb domain in the Group A PfEMP1 protein PF11_0521 (Oleinikov et al., 2009).

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3.8 PfEMP1 and rosetting The ability to form rosettes, which is the binding of multiple uninfected erythrocytes to a central IE, is another long-recognized IE adhesion phenotype, which is not confined to P. falciparum (David et al., 1988; Lowe et al., 1998). In P. falciparum, the ability to form rosettes has been associated with severe disease in many studies, and presence of rosette-inhibitory antibodies has been linked to protection from severe malaria (Carlson et al., 1990; Kun et al., 1998; Rowe et al., 1995; Treutiger et al., 1992). PfEMP1 plays an important role in P. falciparum rosetting, which is a diverse adhesion phenotype that appears to involve multiple PfEMP1 ligands and several erythrocyte receptors (reviewed by Mercereau-Puijalon et al., 2008). The adhesive interaction is mediated by the semi-conserved, N-terminal head structure of PfEMP1 (Chen et al., 1998a, 2000; Ghumra et al., 2012; Rowe et al., 1997). The functional significance of rosetting is unclear. It does not appear to facilitate reinvasion (Clough et al., 1998; Deans et al., 2006), and it may simply be a marker of adhesion to endothelial host receptor motifs also present on erythrocytes. The erythrocyte receptors involved in rosetting appear to be mostly carbohydrates (Barragan et al., 2000; Rowe et al., 1994; Vigan-Womas et al., 2012; Vogt et al., 2004), which would be consistent with such a hypothesis. Moreover, there is a striking correlation between expression of PfEMP1 mediating rosetting and Fc-mediated binding of IgM to the IEs (Rowe et al., 2002; Stevenson et al., in press). The bound IgM was originally proposed to act as ‘bridges’ between the IE and the surrounding erythrocytes (Scholander et al., 1996), but the recent mapping of the IgMbinding site near the C-terminus of PfEMP1 makes this unlikely (Stevenson et al., in press). Instead, it now appears that Fc-dependent binding of IgM e and likely additional soluble serum factors e to rosetting PfEMP1 evolved to overcome the often low affinity between individual PfEMP1 molecules and their carbohydrate receptors by ‘orchestrating’ the adhesive interaction of several PfEMP1 (Stevenson et al., submitted for publication). Whether this would be sufficient to sustain IE adhesion to endothelial carbohydrates by itself, or whether it could constitute one element in adhesion involving additional protein receptors (Adams et al., 2014; Ruangjirachuporn et al., 1991), is currently unknown.

3.9 PfEMP1 and gametocytes At some point during the infection, some intra-erythrocytic parasites leave the asexual multiplication cycle and develop into gametocytes (Figure 3).


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Figure 3 var gene transcription and switching. All var genes are tethered to the periphery of the nucleus (light blue (gray in print versions)) with silent var gene clustered in a repressive centre (light brown (light gray in print versions)) (Lopez-Rubio et al., 2009). (a) During the asexual ring-stage, a single active var gene (green (light gray in print versions)) is transcribed at a perinuclear site with transcription factors (not shown) away from the repressive centre. (bec) At the ring-stage, parasite matures to a trophozoite, but prior to DNA replication, var gene transcription shuts down. However, the ‘active’ var gene remains in a poised state during the trophozoite and sporozoite stages. A putative methyltransferase (PfSET10) has been associated with the transition from the active to the poised state. Whether or not the poised var gene re-enters the repressive centre until reactivation is not known. (d) Free merozoites reinvade uninfected erythrocytes and most of the new ring-stage parasites originating from reinvasion will start transcribing the poised var gene. (e) Some daughter parasites switch transcription from the parental var gene to another var gene. On- and off-rates of different var genes vary and appear to be an intrinsic property of the particular var gene (Horrocks et al., 2004). It is not clear at which point switching occur in the cell cycle. (f) Transition to sexual gametocytes, which do not divide any further. (g) Sporozoite-stage parasites. Within the nucleus (light blue (gray in print versions)), the active var gene is indicated by red (dark gray in print versions) text, the poised var gene by brown (light gray in print versions) text and silenced var genes by black text.

Like late-stage asexual-stage IEs, erythrocytes infected by immature gametocytes sequester in the bone marrow and the spleen, whereas mature (stage V) gametocytes circulate like ring-stage-IEs (Aguilar et al., 2014; Smalley et al., 1981; Thompson and Robertson, 1935). Very young (stages IeIIA)

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gametocyte-IEs have been reported to adhere to CD36 and ICAM-1 in vitro and to express knobs and PfEMP1 (Day et al., 1998; Hayward et al., 1999; Rogers et al., 1996; Smith et al., 2003). This is consistent with an apparent switch to Group C PfEMP1 at gametocytogenesis, regardless of the PfEMP1-type expressed in the preceding asexual cycle (Sharp et al., 2006). However, PfEMP1-independent mechanisms are probably involved in the tissue retention of the more mature noncirculating stages (IIBeIV) of gametocytes, which are knob-less and appear devoid of surface-expressed PfEMP1 (Day et al., 1998; Hayward et al., 1999; Sinden, 1982).

4. STRUCTURAL ORGANIZATION OF PfEMP1 AT THE IE SURFACE Export and display of PfEMP1 on the IE surface is a complex multistep process that depends on motifs in the N-terminal as well as the transmembrane and cytoplasmic domains (Melcher et al., 2010). A detailed review of the molecular details of the transport of PfEMP1 from the parasite to the IE membrane is beyond the scope of this paper, and the reader is referred to several excellent reviews (Boddey and Cowman, 2013; Mundwiler-Pachlatko and Beck, 2013). PfEMP1 can be detected already a few hours after parasite invasion of the erythrocyte, but the proteins do not start to appear on the IE surface until about 16 h post-invasion (Gardner et al., 1996). Surface expression reaches a plateau about 8 h later, and by 30e36 h post-invasion PfEMP1 export to the surface has ceased (Kriek et al., 2003). On the IE surface, PfEMP1 is expressed in clusters on the so-called ‘knobs’, which are membrane protrusions that act as focal points of adhesion between IEs and the endothelium to which they adhere (Luse and Miller, 1971; Miller, 1969). Formation of knobs depends on the parasite knobassociated histidine-rich protein (KAHRP) (Crabb et al., 1997; Kilejian, 1979; Pologe and Ravetch, 1986), which is located on the cytoplasmic side of the IE membrane (Pologe et al., 1987) and anchors the knobs to the cytoskeleton (Leech et al., 1984a; Oh et al., 2000). The conserved intracellular acidic, terminal sequence (ATS) domain of PfEMP1 is also linked to the cytoskeleton. It was originally thought that ATS is bound to KAHRP (Kilejian et al., 1991; Oh et al., 2000; Waller et al., 1999), but more recent data suggest that a member of the parasite-encoded Plasmodium helical interspersed subtelomeric (PHIST) protein family, dubbed lysine-rich membrane-associated PHISTb (LyMP) provides the link between ATS


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and the cytoskeleton (Mayer et al., 2012; Oberli et al., 2014; Proellocks et al., 2014). Disruption of KAHRP results in inability to form knobs, reduced PfEMP1 expression, and decreased IE adhesiveness, although PfEMP1 expression remains clustered (Crabb et al., 1997; Horrocks et al., 2005; Ruangjirachuporn et al., 1991; Schmidt et al., 1982). Although disruption of LyMP causes a similar decrease in IE adhesiveness, both knobs and PfEMP1 expression are retained at wild-type levels (Proellocks et al., 2014). These findings suggest that the PfEMP1 receptor specificity, the topology of the PfEMP1 expression on the IE surface, and their connection to the host cytoskeleton all constitute critical determinant of IE adhesiveness. This conclusion is underpinned by recent findings strongly indicating that haemoglobinopathies such as HbS, HbC and a-thalassemia protect against malaria because P. falciparum-IE adhesion is impaired in hosts carrying these polymorphisms. The ability of the parasites to remodel the cytoskeleton of erythrocytes with these haemoglobin variants is compromised, leading to the formation of abnormal knobs and reduced PfEMP1 expression (Cholera et al., 2008; Cyrklaff et al., 2011; Fairhurst et al., 2005; Krause et al., 2012). It is not yet known how the PfEMP1 proteins are organized on the knobs or even how many are present there, although estimates put the figure in the 10e100 range (Joergensen et al., 2010b). What is known is that the density of knobs increases from their first appearance around 16 h postinvasion until about 35 h post-invasion, after which time-point the density again decreases (Quadt et al., 2012). The knob density also varies among isolates, even when they express the same type of PfEMP1 (Quadt et al., 2012). Finally, it appears that even the PfEMP1 expression on the knobs of a single IE can be heterogeneous, some expressing PfEMP1 while others do not (Horrocks et al., 2005).

5. REGULATION OF VAR GENE TRANSCRIPTION AND SWITCHING Clonal antigenic variation, ensuring the mono-allelic expression of members of the PfEMP1 family, plays a key role in the pathogenesis of P. falciparum malaria and is the central mechanism enabling immune evasion and maintenance of long-term chronic infections (reviewed by Deitsch et al., 2009; Guizetti and Scherf, 2013; Voss et al., 2014). It is therefore of central importance to know how the parasites control transcription of the approximately 60 PfEMP1-encoding var paralogues in the P. falciparum genome, and the switching among them.

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5.1 Transcription of var genes By default, all the var genes in the parasite genome are ‘silenced’ (i.e. not transcribed). This silencing involves several mechanisms, such as reversible histone modifications, promoter-intron ‘pairing’, and tethering of the var genes at the nuclear periphery (Figure 3). Silencing by histone modifications involves two histone deacetylases, Sir2A and Sir2B, which place silencing heterochromatin H3K9me3 marks in the promoter regions of Group A/C/E and Group B var genes, respectively (Duraisingh et al., 2005; Freitas-Junior et al., 2005; Tonkin et al., 2009). H3K9me3 promotes the formation of genetically inactive heterochromatin through recruitment of P. falciparum heterochromatin protein 1 (PfHP1). The importance of this mechanism is illustrated by the observation that disruption of the Sir2 genes causes de-repression of var gene transcription. Silencing due to the interaction between the upstream promoter and noncoding DNA in the intron separating exon 1 and exon 2 of a var gene (Calderwood et al., 2003; Deitsch et al., 2001; Swamy et al., 2011) involves placing of H3K36me3 silencing marks in both exons by the enzymes RNA pol II and PfSET2 (Jiang et al., 2013; Ukaegbu et al., 2014). Interference with the pairing mechanism results in activation of the affected var genes (Avraham et al., 2012; Deitsch et al., 2001; Dzikowski et al., 2006). Most recently, Zhang et al. (2014) identified a chromatin-associated exoribonuclease, PfRNAse II, which specifically silences transcription of Group A var genes. For a var gene to be transcribed, it must be ‘activated’ by releasing it from the silencing mechanisms. Only one of the var genes in the parasite genome is activated and translated into protein during any given 48-h cycle (allelic exclusion), meaning that all the PfEMP1 molecules eventually expressed on the surface of a single IE are identical4. Activation involves removal of repressive histone marks and loss of PfHP1 (Lopez-Rubio et al., 2007; Perez-Toledo et al., 2009) and additional signals mediated by the histone variants H2A.Z andH2B.Z (Hoeijmakers et al., 2013; Petter et al., 2013). It also involves repositioning the activated var gene to an active site distinct from the perinuclear repressive centres (Figure 3) (Lopez-Rubio et al., 2009; Ralph et al., 2005; Voss et al., 2006). Transcription of the active var gene starts shortly after the merozoite invasion of an erythrocyte is completed.

4

A single exception to this rule of mono-allelic exclusion has been reported (Joergensen et al., 2010a). The biological significance of this finding is presently unclear.


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It peaks at the early ring-stage, i.e., about 12 h after invasion, and ceases as the parasites mature to trophozoites about 16 h post-invasion (Chen et al., 1998b; Dahlb€ack et al., 2007; Kyes et al., 2000; Schieck et al., 2007). The formerly active var gene remains in a ‘poised’ state that ensures that the same gene is activated in the next 48-h cycle (epigenetic memory) (Chookajorn et al., 2007; Lopez-Rubio et al., 2007; Volz et al., 2012), except when transcription switches to another var gene.

5.2 Switching transcription from one var gene to another For antigenic variation to work, the parasites must be able to switch from transcription and translation of one var gene to transcribing and translating another (Figure 3). The molecular details of this process remain obscure. Switch rates as high as 2% per generation or more have been reported (Peters et al., 2002; Roberts et al., 1992). The rates of switching to and from individual var genes vary substantially (Fastman et al., 2012; Horrocks et al., 2004) and is affected by chromosomal location (Frank et al., 2007). Switching does not appear to be preprogrammed (‘hard-wired’), but neither does it appear to be completely random (Fastman et al., 2012; Recker et al., 2011). Evidence of external cues that might direct the switching pattern is scarce. As an example, Nunes et al. (2008) did not find evidence that hormones or other soluble indicators of pregnancy induced transcription of var2csa encoding VAR2CSA-type PfEMP1 mediating IE accumulation in the placenta (see Section 6.2). Nevertheless, the spleen somehow modulates IE sequestration, as splenectomy has repeatedly been found to abolish it (Bachmann et al., 2009; David et al., 1983; Hommel et al., 1983). Survival of P. falciparum parasites and transmission from one human host into a new human host require differentiation of asexual parasites into gametocytes. Two recent publications provide evidence that the epigenetics underlying the regulation of var genes is also responsible for regulating the switch from asexual cycling to gametocyte commitment in P. falciparum (Brancucci et al., 2014; Coleman et al., 2014). All var genes are silenced in the sporozoite-stage parasites that are injected during the blood meal of P. falciparum-infected mosquito (Wang et al., 2010), and var gene transcription was found to be highly promiscuous early on in the infection, followed by a marked focussing on a few dominant var genes within a few generations (Lavstsen et al., 2005; Wang et al., 2009). These findings indicate that var gene epigenetic memory is reset during transmission from one host to another, and that the emergence of a dominating var gene at the parasite

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population level is determined to a considerable extent by the fitness of the encoded PfEMP1 for parasite survival in a particular host. This is likely to include the levels of pre-existing immunity and antibodies acquired in the course of an infection, as evidenced by several studies (Cham et al., 2009, 2010; Warimwe et al., 2009).

6. PfEMP1-MEDIATED PATHOGENESIS Many authoritative reviews of P. falciparum malaria pathogenesis are available elsewhere, and this section merely serves to provide an outline of some apparent key processes involving PfEMP1.

6.1 Cerebral malaria Already 120 years ago, Marchiafava and Bignami noted that sequestration of IEs in the capillaries and post-capillary venules of the brain is a characteristic feature of cerebral P. falciparum malaria (Marchiafava and Bignami, 1894). The adhesion of IEs in the cerebral microvasculature involves particular subsets of PfEMP1 with particular receptor specificities (Section 3), and can compromise blood flow and cause inflammation, although the relative importance of these processes is hotly debated (recently reviewed by Storm and Craig, 2014). Some studies have implicated ICAM-1 as an IE receptor involved in the pathogenesis of cerebral malaria (Newbold et al., 1997; Ochola et al., 2011; Turner et al., 1994), but the evidence is far from unequivocal (Heddini et al., 2001; Rogerson et al., 1999). An ICAM-1 polymorphism that occurs at high frequency in Africa does not protect against cerebral malaria (FernandezReyes et al., 1997; Rogerson et al., 1999), and selection of IEs for adhesion to cerebral endothelial cells unexpectedly resulted in expression of DC8- and DC13-type PfEMP1 that do not bind to ICAM-1 (Avril et al., 2012; Claessens et al., 2012). These instead bound to EPCR (Turner et al., 2013). The importance of EPCR as an adhesion receptor of importance in the pathogenesis of severe malaria in general, and cerebral malaria in particular, is supported by findings of perturbed EPCR expression in brain tissue from cerebral malaria patients (Moxon et al., 2013) and of EPCR variants associated with protection from severe malaria, including cerebral malaria (Naka et al., 2014).


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Taken together, these results suggest a pathogenic cascade, where a ‘first wave’ of cerebral IE sequestration mediated by EPCR-binding PfEMP1 (Turner et al., 2013) causes downregulation of EPCR and thrombomodulin (TM) on the endothelium (Moxon et al., 2013). EPCR and TM normally function to control thrombin formation and maintain the integrity of the endothelial barrier through activation of protein C (Stearns-Kurosawa et al., 1996). Expression of EPCR and TM is constitutively low on cerebral endothelium (Laszik et al., 1997), and the EPCR loss induced by the adhering IE appears sufficient to compromise thrombin inactivation by activated protein C, leading to fibrin deposition and breakdown of the endothelial barrier (Moxon et al., 2013; Tripathi et al., 2007). This, and perhaps other pro-inflammatory signals triggered by the adhering IEs, causes upregulation of ICAM-1 (Moxon et al., 2013; Tripathi et al., 2006, 2009), which conceivably leads to sequestration of IEs expressing ICAM-1-binding Group A PfEMP1 (Figure 4). This ‘second wave’ of IE sequestration following the disappearance of EPCR might involve both newly recruited IEs expressing DC4-type PfEMP1 and IEs already there because they express PfEMP1 than can bind to both EPCR and ICAM-1 (see Section 3.2). However, even this complicated scenario may be an oversimplification (Esser et al., 2014), and undoubtedly many details remain to be elucidated.

6.2 Placental malaria As is the case for cerebral malaria, tissue-specific sequestration of IEs is a longrecognized key feature of placental P. falciparum malaria (Blacklock and Gordon, 1925; Clark, 1915). However, the marked concentration of placental malaria among primigravidae remained unexplained until it was realized that the IEs in the placenta have a highly distinct adhesion phenotype that is not compatible with parasite survival in a nonpregnant host (reviewed by Hviid, 2011). By now it is widely accepted that the selective accumulation of IEs in the placenta is mediated by VAR2CSA-type PfEMP1 with affinity for CSA. Although the involvement of other parasite ligands and host receptors has been proposed, the evidence in their favour is scarce. Sequestration of IEs in the placenta is associated with a detrimental proinflammatory response and other disturbances leading to placental insufficiency and intrauterine growth retardation (Fried et al., 1998a; Rogerson et al., 2007; Umbers et al., 2011). However, the specific role of VAR2CSA-type PfEMP1 in these processes remains unclear.

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Figure 4 Linking endothelial protein C receptor (EPCR-) and ICAM-1-binding in cerebral malaria. (a) Effects of EPCR in the absence of P. falciparum-infected erythrocytes (IEs). EPCR plays a crucial role in turning protein C into activated protein C (APC). The APC proteins use EPCR as a co-receptor for cleavage of proteinase-activated receptor 1 (PAR1). The EPCR-APC activation of PAR1 inhibits the nuclear factor-kB pathway and exerts anti-inflammatory and anti-apoptotic activity, and results in protection of endothelial barrier integrity (Stearns-Kurosawa et al., 1996). (b) IEs with surface expression of domain cassette 8 (DC8)- or DC13-type P. falciparum erythrocyte membrane protein 1 (PfEMP1) bind to EPCR. This leads to activation of endothelial cells and release of pro-inflammatory cytokines such as interleukin 1 (IL-1) and tumour necrosis factor a (TNFa), which induce shedding of EPCR and thrombomodulin (TM) from the endothelial surface and increased expression of ICAM-1. The binding of IE expressing DC8- and DC13-type PfEMP1 results in reduced levels of APC and increased thrombin generation and fibrin deposition. The increased thrombin shifts the PAR1 response towards activation of the RhoA and NFkB pathways with induction of ICAM-1 on the endothelial surface. Parasites expressing a DC4-type PfEMP1 or a non-DC4-type PfEMP1 with a shared Duffy-binding-like b (DBLb) ICAM-1 motif subsequently adhere to ICAM-1. A major proportion of such ICAM-1-binding PfEMP1 might initially bind EPCR via their cysteine-rich interdomain region a1.1 (CIDRa1.1), CIDRa1.4, CIDRa1.5 or CIDRa1.7 domains (indicated by a *), and might thus have an intrinsic capacity to induce ICAM-1 expression.


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7. PfEMP1-SPECIFIC IMMUNE RESPONSES Protective immunity to P. falciparum malaria acquired as a result of natural exposure appears to rely heavily on IgG specific for antigens on the erythrocytes infected by the asexual blood stages of the parasite (Cohen et al., 1961). Most of these antigens are highly polymorphic and many, including PfEMP1, are encoded by multigene families (Chan et al., 2014). This polymorphism, in combination with the mono-allelic expression of PfEMP1 and clonal antigenic variation through switching among var genes (Section 5), probably goes a long way towards explaining the long duration of untreated P. falciparum infections (Collins and Jeffery, 1999a,b; Staalsoe et al., 2002) and the slow acquisition of protective immunity in people living in areas with stable transmission of P. falciparum parasites (reviewed by Hviid, 2005). Variant- (largely PfEMP1-) specific protective immunity can also explain why malaria is often seasonal even though parasites are present continuously (Lines and Armstrong, 1992), why not all infections cause disease (Bull et al., 1998; Marsh and Howard, 1986), why protection from complicated disease is acquired first (Bull et al., 2000; Cham et al., 2009; Nielsen et al., 2002), and why young women become sick from malaria (almost) only when they get pregnant for the first time (Fried et al., 1998b; Ricke et al., 2000; Staalsoe et al., 2004). The central importance of PfEMP1, both for the parasites (as these antigens are vital for their survival) and for the host (as they are centrally involved in the virulence of P. falciparum malaria), has created an evolutionary arms race between an immune system trying to control the infection and parasites trying to foil these attempts. As expected in such a scenario, P. falciparum has evolved numerous ways of dodging and sabotaging acquired immunity. Interference with antigen presentation (Urban et al., 1999), antigen camouflaging (Barfod et al., 2011), subversion of immunological memory (Weiss et al., 2009) are some of the mechanisms proposed, in addition to antigenic variation per se. The human host has responded in various ways to these challenges. As an example, it appears likely that several haemoglobinopathies have evolved as a response to the challenge posed by PfEMP1-expressing malaria parasites (Cyrklaff et al., 2012; Fairhurst et al., 2005).

8. PfEMP1 AND VACCINATION AGAINST MALARIA Studies have identified PfEMP1 types (e.g. VAR2CSA, DC4, DC8 and DC13) that are linked to the pathogenesis of malaria on the one

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hand, and shared by many P. falciparum clones on the other (Bengtsson et al., 2013; Lavstsen et al., 2012; Salanti et al., 2003, 2004; Turner et al., 2013). These types therefore constitute potential vaccine targets (Hviid, 2011). VAR2CSA is preferentially expressed by placental parasites and levels of VAR2CSA domain-specific antibodies increase with the number of pregnancies and are associated with reduced placental infections with P. falciparum (Oleinikov et al., 2007; Salanti et al., 2003; Tuikue Ndam et al., 2005; Tuikue Ndam et al., 2006; Tutterrow et al., 2012a,b). These properties have positioned VAR2CSA as a leading pregnancy malaria vaccine candidate and preclinical evaluation comparing the functional activity of anti-adhesive antibodies elicited by different VAR2CSA domains has been undertaken (Fried et al., 2013). The antigenic diversity of non-VAR2CSA PfEMP1 domains and the evidence of promiscuous endothelial cell receptor adhesion, in particular in severe malaria including cerebral malaria, could pose obstacles to the development of an anti-disease vaccine (Esser et al., 2014). It is thus likely, that an effective vaccine against non-placental malaria will require inclusion of non-PfEMP1 antigens, including antigens from merozoites and sporozoites that could supplement immunity to IE surface antigens. A recent attractive candidate of the former type is the merozoite antigen P. falciparum reticulocyte-binding protein homologue 5, which appears essential for merozoite invasion of erythrocytes, and which can induce inhibitory antibodies that are broadly effective (Baum et al., 2009; Bustamante et al., 2012; Douglas et al., 2011; Hayton et al., 2008). Regarding the latter type, the circumsporozoite protein is the current leading candidate (Arama and Troye-Blomberg, 2014; Regules et al., 2011).

9. CONCLUSIONS AND FUTURE DIRECTIONS The understanding of how P. falciparum parasites accomplish the conflicting goals of using PfEMP1 as adhesive proteins binding to a limited set of host receptors on the one hand, and protecting them from immune attack through clonal antigenic variation on the other, is improving at great speed. As an example, new data show that adhesive specificity can be maintained by PfEMP1 proteins having very high sequence diversity (Lau et al., 2015). That finding brings similar studies of trypanosome variant surface glycoproteins to mind (Blum et al., 1993), and calls for detailed studies of T-cell responses to PfEMP1 antigens, a research area that has remained relatively


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unexplored so far (Gitau et al., 2012, 2014). The research community is also eagerly awaiting the results of the comparative analysis of full genome sequences of many P. falciparum clones, which are likely to advance PfEMP1 research dramatically. The first clinical trials of a PfEMP1-based vaccine against placental malaria will start in 2015, and hopes are high (in some quarters, at least) that this and other PfEMP1-based vaccines could act as important gap-fillers in the quest to control, eliminate, and ultimately eradicate the ancient and recalcitrant scourge of malaria.

ACKNOWLEDGEMENTS LH dedicates his contribution to this paper to the late Dr Charlotte Behr, a great colleague and a deeply missed friend. Research in the authors’ laboratories is supported by Augustinus Fonden, The Consultative Committee for Development Research, Danish Council for Independent Research, European Community’s Seventh Framework Programme, Gangsted Fonden, Hørslev Fonden, Novo Nordisk Fonden, Svend Andersen Fonden, and the University of Copenhagen UCPH2016.

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