VSA are important targets of naturally acquired immunity

An early demonstration of the importance of VSA as immune targets was a serological study showing that, among seven different anti-blood stage assays including one measuring opsonization of infected erythrocytes, the titre in children's serum of antibodies that agglutinated infected erythrocytes from a single donor, was the only assay associated with reduced future experience of clinical malaria (Marsh et al. Reference Marsh, Otoo, Hayes, Carson and Greenwood1989). This apparent protection by anti-VSA antibodies against future disease is supported by more recent longitudinal studies by Dodoo et al. (Reference Dodoo, Staalsoe, Giha, Kurtzhals, Akanmori, Koram, Dunyo, Nkrumah, Hviid and Theander2001), where antibodies to infected erythrocytes of 2/5 parasite isolates showed a clear association with protection after age correction. Measures of antibodies to the infected erythrocyte surface present at the time of infection also show a negative association with the severity of disease, supporting a role in immunity (Tebo et al. Reference Tebo, Kremsner, Piper and Luty2002; Yone et al. Reference Yone, Kremsner and Luty2005). Consistent with this, chronic asymptomatic infections are associated with particularly high levels of antibodies to VSA and individuals who carry asymptomatic infections but do not make such responses tend to be more susceptible to future clinical malaria (Bull et al. Reference Bull, Lowe, Kaleli, Njuga, Kortok, Ross, Ndungu, Snow and Marsh2002; Kinyanjui et al. Reference Kinyanjui, Mwangi, Bull, Newbold and Marsh2004; Mackintosh et al. Reference Mackintosh, Mwangi, Kinyanjui, Mosobo, Pinches, Williams, Newbold and Marsh2008). The third approach used in early epidemiological studies was to test for carriage of antibodies that reverse cytoadhesion. The rosetting phenotype, defined as the binding of infected erythrocytes to uninfected erythrocytes, provides a good example. Carlson et al. (Reference Carlson, Helmby, Hill, Brewster, Greenwood and Wahlgren1990) found an association between the presence in children's serum of antibodies that reverse rosetting of a laboratory-adapted parasite line and (1) reduced rosetting in the children's own parasites and (2) absence of cerebral malaria. Antigen variants expressed on the surface of parasites from children with malaria was later shown to correspond with gaps in the repertoire of antibodies carried by the infected individual before they became ill (Bull et al. Reference Bull, Lowe, Kortok, Molyneux, Newbold and Marsh1998; Giha et al. Reference Giha, Staalsoe, Dodoo, Roper, Satti, Arnot, Hviid and Theander2000). Together, these data support a model of naturally acquired immunity to malaria through the gradual acquisition of many variant specific antibodies to VSA.

Such a model is consistent with the known dynamics of P. falciparum infections through information gathered during the time when malaria was used in the therapy of neurosyphilis (Whitrow, Reference Whitrow1990; Karamanou et al. Reference Karamanou, Liappas, Antoniou, Androutsos and Lykouras2013). The importance of VSA as targets of naturally acquired immunity to malaria is highlighted by the observation of clear recrudescence in parasitaemia followed by rapid decline. When these recrudescence occured, they did so with relatively consistent initial growth rates suggesting that new variants were emerging and were subsequently controlled on an otherwise only gradually changing immunological background (Molineaux et al. Reference Molineaux, Diebner, Eichner, Collins, Jeffery and Dietz2001).

Plasmodium falciparum encodes several multi-gene families including var, rif and stevor for which there is evidence of expression on the surface of parasite-infected erythrocytes (Baruch et al. Reference Baruch, Pasloske, Singh, Bi, Ma, Feldman, Taraschi and Howard1995; Smith et al. Reference Smith, Chitnis, Craig, Roberts, Hudson-Taylor, Peterson, Pinches, Newbold and Miller1995; Su et al. Reference Su, Heatwole, Wertheimer, Guinet, Herrfeldt, Peterson, Ravetch and Wellems1995; Cheng et al. Reference Cheng, Cloonan, Fischer, Thompson, Waine, Lanzer and Saul1998; Fernandez et al. Reference Fernandez, Hommel, Chen, Hagblom and Wahlgren1999; Kyes et al. Reference Kyes, Rowe, Kriek and Newbold1999; Niang et al. Reference Niang, Bei, Madnani, Pelly, Dankwa, Kanjee, Gunalan, Amaladoss, Yeo, Bob, Malleret, Duraisingh and Preiser2014; Goel et al. Reference Goel, Palmkvist, Moll, Joannin, Lara, Akhouri, Moradi, Ojemalm, Westman, Angeletti, Kjellin, Lehtio, Blixt, Idestrom, Gahmberg, Storry, Hult, Olsson, von Heijne, Nilsson and Wahlgren2015). Rif and stevor together with the majority of the 60 genes that make up the var gene family are located in the highly diverse sub-telomeric regions of chromosomes (Gardner et al. Reference Gardner, Hall, Fung, White, Berriman, Hyman, Carlton, Pain, Nelson, Bowman, Paulsen, James, Eisen, Rutherford, Salzberg, Craig, Kyes, Chan, Nene, Shallom, Suh, Peterson, Angiuoli, Pertea, Allen, Selengut, Haft, Mather, Vaidya and Martin2002). Of these families the var genes and the PfEMP1 antigens they encode have been the subject of the most research to date because of the role that PfEMP1 play both in cytoadhesion to many specific host receptors and the evidence for their role as targets of naturally acquired antibodies that undergo clonal antigenic variation.

An early study demonstrated how changes in PfEMP1 expression by a laboratory parasite line correlated with switches in both the antigenic and cytoadhesive properties of the infected erythrocyte surface (Biggs et al. Reference Biggs, Gooze, Wycherley, Wollish, Southwell, Leech and Brown1991). After the var genes had been identified (Baruch et al. Reference Baruch, Pasloske, Singh, Bi, Ma, Feldman, Taraschi and Howard1995; Smith et al. Reference Smith, Chitnis, Craig, Roberts, Hudson-Taylor, Peterson, Pinches, Newbold and Miller1995; Su et al. Reference Su, Heatwole, Wertheimer, Guinet, Herrfeldt, Peterson, Ravetch and Wellems1995), switches in var gene expression were correlated with both antigenic variation and altered ability of the parasites to bind to the host receptor molecule ICAM1 (Smith et al. Reference Smith, Chitnis, Craig, Roberts, Hudson-Taylor, Peterson, Pinches, Newbold and Miller1995). Many known host-cell-binding phenotypes identified in P. falciparum-infected erythrocytes have since been mapped onto regions of PfEMP1, including ICAM1 (Smith et al. Reference Smith, Craig, Kriek, Hudson-Taylor, Kyes, Fagan, Pinches, Baruch, Newbold and Miller2000; Bengtsson et al. Reference Bengtsson, Joergensen, Rask, Olsen, Andersen, Turner, Theander, Hviid, Higgins, Craig, Brown and Jensen2013), CD36 (Baruch et al. Reference Baruch, Ma, Singh, Bi, Pasloske and Howard1997; Smith et al. Reference Smith, Kyes, Craig, Fagan, Hudson-Taylor, Miller, Baruch and Newbold1998; Robinson et al. Reference Robinson, Welch and Smith2003), complement receptor 1(Rowe et al. Reference Rowe, Moulds, Newbold and Miller1997), heparin, blood group A antigen (Vigan-Womas et al. Reference Vigan-Womas, Guillotte, Juillerat, Hessel, Raynal, England, Cohen, Bertrand, Peyrard, Bentley, Lewit-Bentley and Mercereau-Puijalon2012) , Chondroitin sulphate A (Salanti et al. Reference Salanti, Staalsoe, Lavstsen, Jensen, Sowa, Arnot, Hviid and Theander2003), PECAM1 (Berger et al. Reference Berger, Turner, Wang, Petersen, Kraft, Lusingu, Mmbando, Marquard, Bengtsson, Hviid, Nielsen, Theander and Lavstsen2013), IgM (Ghumra et al. Reference Ghumra, Semblat, McIntosh, Raza, Rasmussen, Braathen, Johansen, Sandlie, Mongini, Rowe and Pleass2008) and endothelial protein C receptor (EPCR) (Turner et al. Reference Turner, Lavstsen, Berger, Wang, Petersen, Avril, Brazier, Freeth, Jespersen, Nielsen, Magistrado, Lusingu, Smith, Higgins and Theander2013). Recently, knockdowns of var gene expression in two laboratory lines resulted in almost complete loss of antibody recognition and cytoadhesive properties (Chan et al. Reference Chan, Howell, Reiling, Ataide, Mackintosh, Fowkes, Petter, Chesson, Langer, Warimwe, Duffy, Rogerson, Bull, Cowman, Marsh and Beeson2012), suggesting that PfEMP1 are the dominant antigens expressed on the surface of these laboratory-adapted parasite lines that are recognized by naturally acquired antibodies against infected erythrocytes.

Why is the feasibility of a PfEMP1 vaccine worth exploring?

PfEMP1 clearly plays an important role in the host–parasite interaction. However, PfEMP1 is, on the face of it, perhaps the last group of proteins that you might want to study as a malaria vaccine candidate. Early serological studies show that after an episode of disease, children develop antibodies that are highly specific to the parasites that caused that disease episode, suggesting that, at least the immune-dominant epitopes expressed on the infected erythrocyte surface are extremely diverse (Marsh and Howard, Reference Marsh and Howard1986; Forsyth et al. Reference Forsyth, Philip, Smith, Kum, Southwell and Brown1989; Iqbal et al. Reference Iqbal, Perlmann and Berzins1993; Reeder et al. Reference Reeder, Rogerson, Al-Yaman, Anders, Coppel, Novakovic, Alpers and Brown1994; Bull et al. Reference Bull, Lowe, Kortok and Marsh1999). Though they are encoded by a family of only 60 var genes in every genome, each genome is potentially a drop in the ocean of var diversity within the global parasite population and the potential for immune evasion through influx of new PfEMP1 variants through gene conversion would seem from these data to be immense. Barry et al. (Reference Barry, Leliwa-Sytek, Tavul, Imrie, Migot-Nabias, Brown, McVean and Day2007), sampled short tags of sequence from PfEMP1 from a worldwide collection of parasites and failed to find a limit to this sequence diversity. As new sequences were sampled randomly from the ‘pot’ of collected sequences, a point was never reached at which the rate of sampling of new sequences diminished (Barry et al. Reference Barry, Leliwa-Sytek, Tavul, Imrie, Migot-Nabias, Brown, McVean and Day2007). Recent evidence further suggests that var undergo mitotic recombination, potentially unpacking unlimited diversity from a single genome (Claessens et al. Reference Claessens, Hamilton, Kekre, Otto, Faizullabhoy, Rayner and Kwiatkowski2014).

Despite this immense molecular diversity children as they grow up in malaria endemic areas do learn to recognize antigens expressed on the surface of infected erythrocytes (Barragan et al. Reference Barragan, Kremsner, Weiss, Wahlgren and Carlson1998; Bull et al. Reference Bull, Lowe, Kortok, Molyneux, Newbold and Marsh1998) and cross-reactivity exists between epitopes expressed on the surface of infected erythrocytes sampled from different geographical areas and even different continents (Aguiar et al. Reference Aguiar, Albrecht, Cegielski, Greenwood, Jensen, Lallinger, Martinez, McGregor, Minjas, Neequaye, Patarroyo, Sherwood and Howard1992; Nielsen et al. Reference Nielsen, Vestergaard, Lusingu, Kurtzhals, Giha, Grevstad, Goka, Lemnge, Jensen, Akanmori, Theander, Staalsoe and Hviid2004). So why does antigenic variation in P. falciparum not always lead to infections that overwhelm the host? The life cycle of P. falciparum relies on the establishment of chronic blood stage infections and host survival through the dry season when pools of water necessary for mosquito reproduction are scarce and opportunities to transmit to mosquitos is low. The question of how the correct balance is maintained between too little and too much antigenic diversity; host survival and parasite escape from antibodies, is still one of the major questions in malaria parasite biology (Saul, Reference Saul1999). The generally accepted broad explanation is that antigenic diversity in PfEMP1 is constrained by its function in cytoadhering to host cells and bringing about sequestration of infected erythrocytes in tissue capillary beds and that inefficient cytoadhesion leads to passage of the infected erythrocytes through the spleen where they are removed from circulation (Barnwell et al. Reference Barnwell, Howard, Coon and Miller1983).

The idea that the functional role of PfEMP1 constrains its antigenic diversity forms an important part of the rationale for considering PfEMP1 as a viable vaccine target against severe malaria (see Fig. 1). This rationale stems from the way in which naturally acquired immunity develops. Children who grow up in malaria endemic areas probably never develop sterile immunity to P. falciparum. Immunity to severe malaria develops more rapidly than immunity to mild malaria as the host–parasite relationship changes from one associated with severe malaria and transmission to mosquitoes towards one that sustains chronic asymptomatic infections (Langhorne et al. Reference Langhorne, Ndungu, Sponaas and Marsh2008; Goncalves et al. Reference Goncalves, Huang, Morrison, Holte, Kabyemela, Prevots, Fried and Duffy2014; Griffin et al. Reference Griffin, Hollingsworth, Reyburn, Drakeley, Riley and Ghani2015). If constraints on PfEMP1 structure are imposed by effective cytoadhesion and avoidance of the host spleen, this could result in trade-off between antigenic novelty on the one hand and highly effective cytoadhesion on the other (Bull et al. Reference Bull, Lowe, Kortok and Marsh1999; Nielsen et al. Reference Nielsen, Staalsoe, Kurtzhals, Goka, Dodoo, Alifrangis, Theander, Akanmori and Hviid2002; Jensen et al. Reference Jensen, Magistrado, Sharp, Joergensen, Lavstsen, Chiucchiuini, Salanti, Vestergaard, Lusingu, Hermsen, Sauerwein, Christensen, Nielsen, Hviid, Sutherland, Staalsoe and Theander2004; Frank and Bush, Reference Frank and Bush2007; van Noort et al. Reference van Noort, Nunes, Weedall, Hviid and Gomes2010; Buckee and Recker, Reference Buckee and Recker2012; Severins et al. Reference Severins, Klinkenberg and Heesterbeek2012). If only strongly cytoadhesive PfEMP1 can mediate the level of sequestration that can bring about severe malaria and these variants are more structurally constrained and antigenically conserved, then it is possible to imagine this trade-off setting up a spectrum of variants differentially adapted to efficient cytoadhesion on the one hand and antibody evasion on the other. The observed shift in host–parasite interaction observed in children growing up exposed to malaria parasites could be brought about through rapid acquisition of immunity to the few types that support high levels of cytoadhesion and differential survival of parasites expressing variants that can only support uncomplicated and asymptomatic infections.

Fig. 1. Hypothetical trade-offs between function and antigenic novelty. This figure shows an immunological version of the principle of functional constraint focusing on efficiency of biological functions associated with the host–parasite interaction (y-axis) and the hypothetical antigenic space within which those functions exist (x-axis). Optimal function can only be performed by a narrow range of molecular structures which translates, in the simplest case, to a small antigenic space. Key to virulence is not simply the function itself, but the ability of molecules or systems of molecules to uncouple immunogenicity from function to expand the antigenic space available. Hypothetical lines are drawn to suggest trade-offs between function and antigenic space in for: (i) Measles haemagglutinin (Frank and Bush, Reference Frank and Bush2007), (ii) var2CSA, (iii) group A and DC8 PfEMP1 (Buckee and Recker, Reference Buckee and Recker2012), (iv) group B and C PfEMP1, (vi) HIV gp120 (de Boer and Boerlijst, Reference de Boer and Boerlijst1994). Following from this hypothetical trade-off, parasites with high levels of recognition by antibodies commonly carried by children have been proposed to express PfEMP1 variants with strong cytoadhesive function and exist within a small restricted immunological space. Such hypothetical variants have been called: agglutination frequency high (AF^H (Bull et al. Reference Bull, Kortok, Kai, Ndungu, Ross, Lowe, Newbold and Marsh2000) VSA with a high frequency of recognition [VSA_FoRH (Bull et al. Reference Bull, Pain, Ndungu, Kinyanjui, Roberts, Newbold and Marsh2005b)], and VSA associated with severe malaria [VSA_SM (Nielsen et al. Reference Nielsen, Staalsoe, Kurtzhals, Goka, Dodoo, Alifrangis, Theander, Akanmori and Hviid2002; Jensen et al. Reference Jensen, Magistrado, Sharp, Joergensen, Lavstsen, Chiucchiuini, Salanti, Vestergaard, Lusingu, Hermsen, Sauerwein, Christensen, Nielsen, Hviid, Sutherland, Staalsoe and Theander2004)]. Their position within the graph is indicated with a box

Evidence for this model comes first from the association between various cytoadhesion phenotypes and the severity of malaria (Carlson et al. Reference Carlson, Helmby, Hill, Brewster, Greenwood and Wahlgren1990; Rowe et al. Reference Rowe, Obeiro, Newbold and Marsh1995; Newbold et al. Reference Newbold, Warn, Black, Berendt, Craig, Snow, Msobo, Peshu and Marsh1997; Heddini et al. Reference Heddini, Pettersson, Kai, Shafi, Obiero, Chen, Barragan, Wahlgren and Marsh2001; Ochola et al. Reference Ochola, Siddondo, Ocholla, Nkya, Kimani, Williams, Makale, Liljander, Urban, Bull, Szestak, Marsh and Craig2011; Turner et al. Reference Turner, Lavstsen, Berger, Wang, Petersen, Avril, Brazier, Freeth, Jespersen, Nielsen, Magistrado, Lusingu, Smith, Higgins and Theander2013). The second line of evidence comes from studies that have examined variation in the serological commonness of antigens expressed on the surface of infected erythrocytes measured by panels of plasma or serum. Parasites from children with severe malaria and those who were very young tend to be more commonly recognized (Bull et al. Reference Bull, Lowe, Kortok and Marsh1999, Reference Bull, Kortok, Kai, Ndungu, Ross, Lowe, Newbold and Marsh2000; Nielsen et al. Reference Nielsen, Staalsoe, Kurtzhals, Goka, Dodoo, Alifrangis, Theander, Akanmori and Hviid2002). The fact that severe malaria is a rare manifestation of infection seems at odds with the idea that it is associated with commonly occurring variants and raises the question of why some children get severe malaria and others don't. We might hypothesize that the expression of subsets of commonly circulating PfEMP1 is necessary, if not sufficient, for the development of severe malaria. However, this would be at odds with the observation that parasites from 13/42 children with severe malaria in (Bull et al. Reference Bull, Kortok, Kai, Ndungu, Ross, Lowe, Newbold and Marsh2000) were not agglutinated by any of a panel of 15 children's plasma (Bull et al. Reference Bull, Pain, Ndungu, Kinyanjui, Roberts, Newbold and Marsh2005b). The serological profiles of commonly recognized parasites were also diverse showing that there may be many commonly recognized types circulating in the parasite population (Bull et al. Reference Bull, Lowe, Kortok and Marsh1999, Reference Bull, Kortok, Kai, Ndungu, Ross, Lowe, Newbold and Marsh2000, Reference Bull, Pain, Ndungu, Kinyanjui, Roberts, Newbold and Marsh2005b; Nielsen et al. Reference Nielsen, Staalsoe, Kurtzhals, Goka, Dodoo, Alifrangis, Theander, Akanmori and Hviid2002).

PfEMP1 structure reflects the tension between cytoadhesive function and antigenic novelty

The structure of PfEMP1 nicely reflects its dual role as an antigenically variant cytoadhesive molecule. Despite considerable molecular diversity and length variation between the PfEMP1 variants, there is underlying structural conservation at various levels. This can be summarized as follows: (1) the molecules are constructed from only two broad classes of domain [see (Higgins and Carrington, Reference Higgins and Carrington2014) for a recent review]. The most common type of domain, ‘duffy binding-like’ (DBL) exists in several forms in Plasmodium species, in the duffy blood group binding protein in Plasmodium vivax, and in P. falciparum, the EBA140 and EBA175 erythrocyte-binding antigens and in the MSPDBL2 merozoite surface protein. The other domain type found in PfEMP1 is called the ‘cysteine-rich interdomain region’ (CIDR). (2) Both CIDR and DBL contain blocks of sequence (homology blocks) that are relatively conserved. These homology blocks can be used to divide each domain type into various classes and subclasses (Rask et al. Reference Rask, Hansen, Theander, Gorm Pedersen and Lavstsen2010). (3) The key feature of these domain subclasses is that they represent deep divisions within the sequences. These broad classes of domain are commonly encountered between different parasite isolates and antigenic differences found within each parasite genome appear to be broadly conserved (Joergensen et al. Reference Joergensen, Turner, Magistrado, Dahlback, Vestergaard, Lusingu, Lemnge, Salanti, Theander and Jensen2006, Reference Joergensen, Vestergaard, Turner, Magistrado, Lusingu, Lemnge, Theander and Jensen2007). This is reminiscent of the deep divide seen within MHC genes (Klein and O'Huigin, Reference Klein and O'Huigin1994) and is supported by the existence of a similar range of common sequence features in var sequences from Plasmodium reichenowi, a parasite species similar to P. falciparum that infects chimpanzees (Bull et al. Reference Bull, Buckee, Kyes, Kortok, Thathy, Guyah, Stoute, Newbold and Marsh2008; Zilversmit et al. Reference Zilversmit, Chase, Chen, Awadalla, Day and McVean2013; Otto et al. Reference Otto, Rayner, Bohme, Pain, Spottiswoode, Sanders, Quail, Ollomo, Renaud, Thomas, Prugnolle, Conway, Newbold and Berriman2014). In the case of PfEMP1 domains, specific cytoadhesive phenotypes are associated with specific domain subtypes (Fig. 2 and Table 1). (4) Finally, the predicted three-dimensional structures of these domains tends to be conserved and PfEMP1 molecules associated with childhood malaria are thought to exist as loose modular string of domains each with one of two basic structures (Klein et al. Reference Klein, Gittis, Su, Makobongo, Moore, Singh, Miller and Garboczi2008; Higgins and Carrington, Reference Higgins and Carrington2014).

Fig. 2. The structures of some notable PfEMP1 variants. Duffy-binding domains (DBL) are shown as rectangles and cysteine-rich interdomain regions (CIDR) and the CIDR-like domain of var2CSA are shown as ovals, N-terminal segment and acidic terminal segment (ATS) sequences are shown as hatched and stippled squares, respectively. ID2a and ID2b are interdomain regions within var2CSA that from part of the DBL2X CSA-binding domain (Clausen et al. Reference Clausen, Christoffersen, Dahlback, Langkilde, Jensen, Resende, Agerbaek, Andersen, Berisha, Ditlev, Pinto, Nielsen, Theander, Larsen and Salanti2012). Cytoadhesion phenotypes associated with specific domains are shown as colours. Recombinant proteins derived from these PfEMP1 are shown as black horizontal lines under the region of the molecules from which they were derived a, b, c, d, e, f. Domain cassettes are shown as horizontal dashed lines: a, the Id1-DBL2X region can stimulate antibodies in mice that inhibit CSA binding (Bordbar et al. Reference Bordbar, Tuikue-Ndam, Bigey, Doritchamou, Scherman and Deloron2012); b, The r179 region of CD36-binding CIDR from the Malayan Camp line used by Baruch et al. (Reference Baruch, Ma, Singh, Bi, Pasloske and Howard1997) to induce homologous protection in Aotus monkeys; c, the region of the IgM binding, rosette mediating TM284var1 used by Ghumra et al. (Reference Ghumra, Semblat, Ataide, Kifude, Adams, Claessens, Anong, Bull, Fennell and Arman2012) to induce cross-reactive, opsonizing antibodies in rabbits; IT4var60 was found to be the rosette mediating var in the well-studied cell line FCR3S1·2 (Albrecht et al. Reference Albrecht, Moll, Blomqvist, Normark, Chen and Wahlgren2011); d,e, constructs from non-IgM-binding, rosette-mediating PfEMP1 found to induce non-cross-reactive, rosette-inhibiting antibodies (Vigan-Womas et al. Reference Vigan-Womas, Guillotte, Juillerat, Vallieres, Lewit-Bentley, Tall, Baril, Bentley and Mercereau-Puijalon2011); (Ghumra et al. Reference Ghumra, Semblat, Ataide, Kifude, Adams, Claessens, Anong, Bull, Fennell and Arman2012); The R29 line was recently found to simultaneously form rosettes and bind to human brain endothelial cells (Adams et al. Reference Adams, Kuhnrae, Higgins, Ghumra and Rowe2014); f, naturally acquired antibodies to the DBLβ4 domain of pf11_0008 were associated with protection from future malaria in a longitudinal study (Magistrado et al. Reference Magistrado, Lusingu, Vestergaard, Lemnge, Lavstsen, Turner, Hviid, Jensen and Theander2007). A PECAM1-binding domain cassette (DC5) was subsequently identified within this gene (Berger et al. Reference Berger, Turner, Wang, Petersen, Kraft, Lusingu, Mmbando, Marquard, Bengtsson, Hviid, Nielsen, Theander and Lavstsen2013). Also shown are DCs, DC4 found to bind ICAM1 (Bengtsson et al. Reference Bengtsson, Joergensen, Rask, Olsen, Andersen, Turner, Theander, Hviid, Higgins, Craig, Brown and Jensen2013), identified within var4 previously found to be dominantly expressed in parasites selected for binding to antibodies from semi-immune children (Jensen et al. Reference Jensen, Magistrado, Sharp, Joergensen, Lavstsen, Chiucchiuini, Salanti, Vestergaard, Lusingu, Hermsen, Sauerwein, Christensen, Nielsen, Hviid, Sutherland, Staalsoe and Theander2004). Note the additional presence of a CIDRα1.6 within var4, shown to bind to EPCR by Lau et al. (Reference Lau, Turner, Jespersen, Lowe, Petersen, Wang, Petersen, Lusingu, Theander, Lavstsen and Higgins2015) but not by Turner et al. (Reference Turner, Lavstsen, Berger, Wang, Petersen, Avril, Brazier, Freeth, Jespersen, Nielsen, Magistrado, Lusingu, Smith, Higgins and Theander2013). IT4var6 and IT4var19 were two genes selected for binding to cells lines derived from human brain endothelial cells (Avril et al. Reference Avril, Tripathi, Brazier, Andisi, Janes, Soma, Sullivan, Bull, Stins and Smith2012; Claessens et al. Reference Claessens, Adams, Ghumra, Lindergard, Buchan, Andisi, Bull, Mok, Gupta and Wang2012), within which the DC8 and the EPCR-binding CIDRa1·1 domain was identified (Turner et al. Reference Turner, Lavstsen, Berger, Wang, Petersen, Avril, Brazier, Freeth, Jespersen, Nielsen, Magistrado, Lusingu, Smith, Higgins and Theander2013).

Table 1. Immunological studies on PfEMP1 domains

DC, domain cassette; SD, sub-domain; HB, homology block; var, commonly used model var; SM, association between expression and disease severity; C/R Ab, Whether cross-reactive antibodies have been detected; P. reich., identification of structure in Plasmodium reichenowi; antibodies, source of antibodies used; protection, the method used to demonstrate that antibodies are protective; longitudinal, longitudinal cohort studies of human populations; adhesion, reversal of infected erythrocyte adhesion; vaccination, protection demonstrated by vaccinating animals; anti-roset, rosette disruption by antibodies. RD, malaria with respiratory distress.

References: 1. Ghumra et al. (Reference Ghumra, Semblat, Ataide, Kifude, Adams, Claessens, Anong, Bull, Fennell and Arman2012); 2. Stevenson et al. (Reference Stevenson, Huda, Jeppesen, Laursen, Rowe, Craig, Streicher, Barfod and Hviid2014); 3. Berger et al. (Reference Berger, Turner, Wang, Petersen, Kraft, Lusingu, Mmbando, Marquard, Bengtsson, Hviid, Nielsen, Theander and Lavstsen2013); 4. Jensen et al. (Reference Jensen, Magistrado, Sharp, Joergensen, Lavstsen, Chiucchiuini, Salanti, Vestergaard, Lusingu, Hermsen, Sauerwein, Christensen, Nielsen, Hviid, Sutherland, Staalsoe and Theander2004); 5. Bengtsson et al. (Reference Bengtsson, Joergensen, Rask, Olsen, Andersen, Turner, Theander, Hviid, Higgins, Craig, Brown and Jensen2013); 6. Lau et al. (Reference Lau, Turner, Jespersen, Lowe, Petersen, Wang, Petersen, Lusingu, Theander, Lavstsen and Higgins2015); 7. Janes et al. (Reference Janes, Wang, Levin-Edens, Vigan-Womas, Guillotte, Melcher, Mercereau-Puijalon and Smith2011); 8. Avril et al. (Reference Avril, Tripathi, Brazier, Andisi, Janes, Soma, Sullivan, Bull, Stins and Smith2012); 9. Claessens et al. (Reference Claessens, Adams, Ghumra, Lindergard, Buchan, Andisi, Bull, Mok, Gupta and Wang2012); 10. Lavstsen et al. (Reference Lavstsen, Turner, Saguti, Magistrado, Rask, Jespersen, Wang, Berger, Baraka, Marquard, Seguin-Orlando, Willerslev, Gilbert, Lusingu and Theander2012); 11. Turner et al. (Reference Turner, Lavstsen, Berger, Wang, Petersen, Avril, Brazier, Freeth, Jespersen, Nielsen, Magistrado, Lusingu, Smith, Higgins and Theander2013); 12. Gamain et al. (Reference Gamain, Miller and Baruch2001); 13. Magistrado et al. (Reference Magistrado, Lusingu, Vestergaard, Lemnge, Lavstsen, Turner, Hviid, Jensen and Theander2007); 14. Vigan-Womas et al. (Reference Vigan-Womas, Guillotte, Juillerat, Vallieres, Lewit-Bentley, Tall, Baril, Bentley and Mercereau-Puijalon2011); 15. Vigan-Womas et al. (Reference Vigan-Womas, Guillotte, Juillerat, Hessel, Raynal, England, Cohen, Bertrand, Peyrard, Bentley, Lewit-Bentley and Mercereau-Puijalon2012); 16. Angeletti et al. (Reference Angeletti, Albrecht, Blomqvist, Quintana Mdel, Akhter, Bachle, Sawyer, Sandalova, Achour, Wahlgren and Moll2012); 17. Albrecht et al. (Reference Albrecht, Angeletti, Moll, Blomqvist, Valentini, D'Alexandri, Maurer and Wahlgren2014); 18. Normark et al. (Reference Normark, Nilsson, Ribacke, Winter, Moll, Wheelock, Bayarugaba, Kironde, Egwang, Chen, Andersson and Wahlgren2007); 19. Blomqvist et al. (Reference Blomqvist, Albrecht, Quintana Mdel, Angeletti, Joannin, Chene, Moll and Wahlgren2013); 20. Patarroyo et al. (Reference Patarroyo, Alba, Curtidor, Vanegas, Almonacid and Patarroyo2014).

Functional specialization is supported by serological and expression studies

A single clear example shows how effectively evolution has shaped the PfEMP1 repertoire to adapt both immunologically and functionally to its host. Malaria in pregnancy is associated with a single PfEMP1 variant encoded by var2CSA (Salanti et al. Reference Salanti, Staalsoe, Lavstsen, Jensen, Sowa, Arnot, Hviid and Theander2003, see Box 1). This PfEMP1 binds specifically to chondroitin sulphate A but not to CD36 and is adapted to bind to syncitiotrophoblasts in the placenta. Parasites isolated from placenta are poorly recognized by antibodies from children, male adults and women during their first pregnancy but immunity develops during subsequent pregnancies, explaining the observed parity dependency in susceptibility to placental malaria (Beeson et al. Reference Beeson, Brown, Molyneux, Mhango, Dzinjalamala and Rogerson1999).

This functional specialization shapes the entire PfEMP1 repertoire. PfEMP1 associated with childhood malaria can be divided into three broad groups, A, B and C defined by upstream (ups) elements A, B and C, respectively (Lavstsen et al. Reference Lavstsen, Salanti, Jensen, Arnot and Theander2003). Unlike the majority of var genes, those in group C are located in clusters close to centromeres. Diversity of var genes is maintained by gene conversion events that occur between non-homologous positions within the genome. As a result the architectures of var genes, that is, the combinations and number of various DBL and CIDR domains, are highly variable between genomes (Kraemer and Smith, Reference Kraemer and Smith2003). Despite this, the group A genes appear to be relatively genetically isolated from the B and C genes. Group A PfEMP1 also tend to be longer and contain more DBL and CIDR domains. In contrast, there is evidence for considerable gene conversion between B and C var genes (Kraemer and Smith, Reference Kraemer and Smith2003; Kraemer et al. Reference Kraemer, Kyes, Aggarwal, Springer, Nelson, Christodoulou, Smith, Wang, Levin, Newbold, Myler and Smith2007; Rask et al. Reference Rask, Hansen, Theander, Gorm Pedersen and Lavstsen2010).

Expression of group A PfEMP1 by the infecting parasite population tends to be associated with infections of children with low immunity. This class of PfEMP1 tends to be recognized first as children are exposed to malaria parasites during the first few years of their lives. Parasites selected in vitro for recognition by pooled children's antibodies have a tendency to express Group A PfEMP1 (Jensen et al. Reference Jensen, Magistrado, Sharp, Joergensen, Lavstsen, Chiucchiuini, Salanti, Vestergaard, Lusingu, Hermsen, Sauerwein, Christensen, Nielsen, Hviid, Sutherland, Staalsoe and Theander2004). Recombinant domains from PfEMP1 are recognized in a hierarchical manner and group A PfEMP1 are the first to be recognized (Cham et al. Reference Cham, Turner, Lusingu, Vestergaard, Mmbando, Kurtis, Jensen, Salanti, Lavstsen and Theander2009, Reference Cham, Turner, Kurtis, Mutabingwa, Fried, Jensen, Lavstsen, Hviid, Duffy and Theander2010). Finally, parasite expression of group A-like PfEMP1 is differentially selected against in vivo as children develop antibodies against the IE surface (Bull et al. Reference Bull, Berriman, Kyes, Quail, Hall, Kortok, Marsh and Newbold2005a; Rottmann et al. Reference Rottmann, Lavstsen, Mugasa, Kaestli, Jensen, Muller, Theander and Beck2006; Warimwe et al. Reference Warimwe, Keane, Fegan, Musyoki, Newton, Pain, Berriman, Marsh and Bull2009, Reference Warimwe, Fegan, Musyoki, Newton, Opiyo, Githinji, Andisi, Menza, Kitsao, Marsh and Bull2012) (see Table 2). In contrast, the expression of group C var genes tends, at least in children from Papua New Guinea, to be differentially elevated in children with asymptomatic infections (Kaestli et al. Reference Kaestli, Cockburn, Cortes, Baea, Rowe and Beck2006; Falk et al. Reference Falk, Kaestli, Qi, Ott, Baea, Cortes and Beck2009).

Table 2. A summary of PfEMP1 expression studies in clinical parasite isolates

Positive and negative associations are shown as + and − symbol, respectively, lack of association is shown as 0, blank means that the comparison was not done. Four levels of immunity are described: Disease severity refers to comparisons between severe and mild malaria, Asymptomatic refers to comparisons between asymptomatic and symptomatic infection; age refers to whether there was an association with higher age of the parasite donor at the time of infection; antibodies asks whether there was an association between var expression and levels of antibodies to infected erythrocytes. Var expression is described as A, Group A; B, group B; C, group C; 8, DC8; 13, DC13; H, Homogenetiy of the var expression profile i.e. the extent to which a single variant dominates. Methods: DBLαtag, an expressed sequence tag approach using cloned and sequenced, PCR amplified tags from the DBLα domain; qPCR, quantitative PCR using domain specific primers; MS, Mass spectrometry; MA, microarray.

References for each study are coded as follows: 1. Kirchgatter and Portillo Hdel (Reference Kirchgatter and Portillo Hdel2002); 2. Kaestli et al. (Reference Kaestli, Cockburn, Cortes, Baea, Rowe and Beck2006); 3. Falk et al. (Reference Falk, Kaestli, Qi, Ott, Baea, Cortes and Beck2009); 4. Bull et al. (Reference Bull, Berriman, Kyes, Quail, Hall, Kortok, Marsh and Newbold2005a); 5. Warimwe et al. (Reference Warimwe, Keane, Fegan, Musyoki, Newton, Pain, Berriman, Marsh and Bull2009); 6. Warimwe et al. (Reference Warimwe, Fegan, Musyoki, Newton, Opiyo, Githinji, Andisi, Menza, Kitsao, Marsh and Bull2012); 7. Warimwe et al. (Reference Warimwe, Fegan, Musyoki, Newton, Opiyo, Githinji, Andisi, Menza, Kitsao, Marsh and Bull2013); 8. Rottmann et al. (Reference Rottmann, Lavstsen, Mugasa, Kaestli, Jensen, Muller, Theander and Beck2006); 9. Lavstsen et al. (Reference Lavstsen, Turner, Saguti, Magistrado, Rask, Jespersen, Wang, Berger, Baraka, Marquard, Seguin-Orlando, Willerslev, Gilbert, Lusingu and Theander2012); 10. Tembo et al. (Reference Tembo, Nyoni, Murikoli, Mukaka, Milner, Berriman, Rogerson, Taylor, Molyneux, Mandala, Craig and Montgomery2014); 11. Normark et al. (Reference Normark, Nilsson, Ribacke, Winter, Moll, Wheelock, Bayarugaba, Kironde, Egwang, Chen, Andersson and Wahlgren2007); 12. Kyriacou et al. (Reference Kyriacou, Stone, Challis, Raza, Lyke, Thera, Kone, Doumbo, Plowe and Rowe2006); 13. Kalmbach et al. (Reference Kalmbach, Rottmann, Kombila, Kremsner, Beck and Kun2010); 14. Merrick et al. (Reference Merrick, Huttenhower, Buckee, Amambua-Ngwa, Gomez-Escobar, Walther, Conway and Duraisingh2012); 15. Bertin et al. (Reference Bertin, Lavstsen, Guillonneau, Doritchamou, Wang, Jespersen, Ezimegnon, Fievet, Alao, Lalya, Massougbodji, Ndam, Theander and Deloron2013); 16. Almelli et al. (Reference Almelli, Nuel, Bischoff, Aubouy, Elati, Wang, Dillies, Coppee, Ayissi, Basco, Rogier, Ndam, Deloron and Tahar2014).

This antigenic separation between group A and non-group A PfEMP1 reflects a functional difference between these two broad classes. Recombinant CIDR domains from the ‘head structure’ (i.e. the first DBL and CIDR domain) of non-group A PfEMP1 molecules usually bind to CD36, while those from group A PfEMP1 do not (Robinson et al. Reference Robinson, Welch and Smith2003; Janes et al. Reference Janes, Wang, Levin-Edens, Vigan-Womas, Guillotte, Melcher, Mercereau-Puijalon and Smith2011). Recently, the phenomenon of mitotic recombination that would lead to immense additional PfEMP1 diversity appears to be restricted to non-group A PfEMP1 (Claessens et al. Reference Claessens, Hamilton, Kekre, Otto, Faizullabhoy, Rayner and Kwiatkowski2014). Together these observations are consistent with the idea of a trade-off between function and antigenic novelty shaping the genomic organization of the var gene family.

Exceptions to the molecular ‘rules’ may enable parasites to evade antibodies, prolong infections and cause severe malaria

For the sake of clarity, we have greatly simplified the distinction made above between the three classes of PfEMP1 types A, B and C. There are clear exceptions to these classifications and these exceptions to the norm may be of importance to our understanding of how these molecules evade antibodies. A transitional group between A and B PfEMP1 genes (B/A) exist that have upsB promotors but are otherwise like group A genes (Lavstsen et al. Reference Lavstsen, Salanti, Jensen, Arnot and Theander2003) and are predicted not to bind CD36 (Robinson et al. Reference Robinson, Welch and Smith2003). Similarly transitional genes between groups B & C (B/C) are common (Lavstsen et al. Reference Lavstsen, Salanti, Jensen, Arnot and Theander2003). The existence of transitional groups indicates that the three classes of genes, cannot be considered as genetically distinct families of molecules.

To refine our understanding of var gene organization, Rask et al. (Reference Rask, Hansen, Theander, Gorm Pedersen and Lavstsen2010) developed a system of classification that works from the level of individual homology blocks and domain subclasses to define common structures within var genes independent of their upstream promotors. This has greatly expanded our ability to describe similarity and differences between PfEMP1 molecules. The most immediately useful idea has been that of the ‘domain cassette’ (DC). The most prominent DCs are shown in Fig. 2 and Table 1. Two DCs in particular, DC8 and DC13 are of interest because genes containing these were found to be enriched by selecting infected erythrocytes for in vitro adhesion to cell lines derived from human brain endothelial cells (Avril et al. Reference Avril, Tripathi, Brazier, Andisi, Janes, Soma, Sullivan, Bull, Stins and Smith2012; Claessens et al. Reference Claessens, Adams, Ghumra, Lindergard, Buchan, Andisi, Bull, Mok, Gupta and Wang2012) and are expressed in children with severe malaria (Lavstsen et al. Reference Lavstsen, Turner, Saguti, Magistrado, Rask, Jespersen, Wang, Berger, Baraka, Marquard, Seguin-Orlando, Willerslev, Gilbert, Lusingu and Theander2012). Specific CIDR domain subclasses found within these DCs, instead of binding to CD36, are associated with binding to EPCR Adhesion to EPCR has been proposed to cause dysregulation of inflammation in the brain leading to cerebral malaria (Moxon et al. Reference Moxon, Wassmer, Milner, Chisala, Taylor, Seydel, Molyneux, Faragher, Esmon, Downey, Toh, Craig and Heyderman2013; Turner et al. Reference Turner, Lavstsen, Berger, Wang, Petersen, Avril, Brazier, Freeth, Jespersen, Nielsen, Magistrado, Lusingu, Smith, Higgins and Theander2013). CIDR domains important to EPCR binding contain conserved epitopes close to the EPCR-binding site that are recognized by malaria-exposed children (Lau et al. Reference Lau, Turner, Jespersen, Lowe, Petersen, Wang, Petersen, Lusingu, Theander, Lavstsen and Higgins2015). In terms of vaccine development, these results are encouraging and further support the idea of broad functional specialization within PfEMP1 family.

Overall these studies demonstrate that modularity exists at various levels within the structure of PfEMP1 associated with childhood malaria: at the level of DCs, domain classes and homology blocks. This would potentially enable different functional and antigenic components to exist in varying contexts. Whether this modularity could delay responses to previously encountered functional domains through re-assortment of T-cell and B-cell epitopes is yet to be explored. Buckee and Recker (Reference Buckee and Recker2012) have suggested that the existence of large numbers of different functional domains on group A PfEMP1 could enable these molecules to support endothelial binding even after antibodies have reversed binding mediated by one or more of the domains. The observation of highly homogeneous expression of group A PfEMP1 in the presence of high levels of antibodies in both severe malaria (Warimwe et al. Reference Warimwe, Recker, Kiragu, Buckee, Wambua, Musyoki, Marsh and Bull2013) and asymptomatic infection (Warimwe et al. Reference Warimwe, Fegan, Musyoki, Newton, Opiyo, Githinji, Andisi, Menza, Kitsao, Marsh and Bull2012) suggests that these antigenically more conserved variants can survive in individuals with high antibody responses, highlighting the need to fully understand how parasites evade antibodies. We have tended to emphasize the rapid acquisition of immunity to severe non-cerebral malaria (Gupta et al. Reference Gupta, Snow, Donnelly, Marsh and Newbold1999b) rather than the fact that cerebral malaria has a slightly later age prevalence than other forms of severe malaria (Greenwood et al. Reference Greenwood, Marsh and Snow1991; Marsh, Reference Marsh1992; Gupta et al. Reference Gupta, Snow, Donnelly and Newbold1999a; Roca-Feltrer et al. Reference Roca-Feltrer, Carneiro, Smith, Schellenberg, Greenwood and Schellenberg2010; Griffin et al. Reference Griffin, Hollingsworth, Reyburn, Drakeley, Riley and Ghani2015) and may involve the ability of parasites to effectively escape from the existing antibody responses (see also Box 2).

Immunological studies of PfEMP1 domains

Five kinds of question need to be addressed in immunological and epidemiological studies to determine the extent to which different features of var genes are targets of naturally acquired immunity to malaria and/or potential vaccine candidates: (1) Are recombinant domains recognized broadly by naturally acquired antibodies or antibodies raised in animals? (2) Do antibodies raised in animals or adsorbed from human serum, bind to the surface of parasite infected erythrocytes, opsonise-infected erythrocytes or reverse cytoadhesion? (3) Is carriage of these antibodies associated with low frequency of future episodes of malaria or low severity of a current infection? (4) Is expression of the PfEMP1 feature by parasites in vivo negatively associated with host immunity measured as disease severity, IE antibodies carried at the time of infection or host age? (5) Are there conserved epitopes in PfEMP1 that can be targeted by antibodies? (6) Can antibodies be shown directly to protect against malaria?

Tables 1 and 2 together with Fig. 2 bring together some of the key information that has been gathered in relation to these questions. Considered alone these questions have important limitations. Question 1 does not distinguish between epitopes exposed by living or dead parasites. Question 3 carries the danger of confounding by cumulative exposure. Because of the diversity and immunogenicity of PfEMP1, recognition of various domains is likely to be excellent markers of past exposure, possibly better than the ‘exposure’ controls used.

Parasite expression levels in association with antibodies or age at the time of disease are likely to be a powerful approach to dissect specific components of the naturally acquired immune response. However, expression studies in parasites still rely heavily on indirect methods. Expression of DCs currently relies on qPCR using sets of primers that identify homology blocks within domains commonly encountered within specific DCs. It is important to note that this approach measures expression levels of specific DCs only indirectly and assumes that strong associations between homology blocks is maintained within the parasite population (Lavstsen et al. Reference Lavstsen, Turner, Saguti, Magistrado, Rask, Jespersen, Wang, Berger, Baraka, Marquard, Seguin-Orlando, Willerslev, Gilbert, Lusingu and Theander2012). Further information is needed on the stability of DCs over time and geographical space. To understand the effect of antibody selection on various different sequence features within var genes we urgently need complete sequence information from expressed var genes from parasites infecting children with different levels of naturally acquired antibodies. This will help us to fully understand why children succumb to life-threatening disease. To this end, a recent study has used mass spectrometry to identify sequence features related to DC8 in children with severe malaria (Bertin et al. Reference Bertin, Lavstsen, Guillonneau, Doritchamou, Wang, Jespersen, Ezimegnon, Fievet, Alao, Lalya, Massougbodji, Ndam, Theander and Deloron2013), but there are still only very few studies that have attempted to place var gene expression profiles at the time of severe malaria within the context of the immune response (Table 2).

A summary of attempts to directly address the challenge of developing a PfEMP1-based vaccine to childhood malaria

In this section, we will summarize the progress that has been made in direct attempts to address question 5 above and protect animals through vaccination with PfEMP1.

Global approaches to finding important protective epitopes on PfEMP1

Using an entirely different approach, Blomqvist et al. (Reference Blomqvist, Albrecht, Quintana Mdel, Angeletti, Joannin, Chene, Moll and Wahlgren2013) used var gene expression data of parasites from Ugandan children to identify short stretches of PfEMP1 sequence that are associated with severe malaria (Normark et al. Reference Normark, Nilsson, Ribacke, Winter, Moll, Wheelock, Bayarugaba, Kironde, Egwang, Chen, Andersson and Wahlgren2007). They raised antibodies to peptides based around these motifs. One of these peptides called RDSM (respiratory distress severe malaria) containing the ALNRKE motif and associated with respiratory distress, stimulated production of antibodies that cross-reacted against several parasite lines including R29, whose expressed var gene (IT4 var9) contains the very similar AINRKE motif (Blomqvist et al. Reference Blomqvist, Albrecht, Quintana Mdel, Angeletti, Joannin, Chene, Moll and Wahlgren2013). This region is in a relatively conserved part of the molecule in the same region as another peptide from var2CSA that is associated with changes in sequence expression between primigravid and multigravid women (Dahlback et al. Reference Dahlback, Rask, Andersen, Nielsen, Ndam, Resende, Turner, Deloron, Hviid, Lund, Pedersen, Theander and Salanti2006).

Future directions

There have been some exciting recent developments in our understanding of the PfEMP1 family of surface antigens, but there are clearly challenges ahead.

Despite the plausible mechanism that would explain the pathology of cerebral malaria (Taylor et al. 2013) it is still unclear whether EPCR is a common receptor for parasites since only a relatively small number of clinical parasite isolates have so far been tested. A recent paper by Esser et al. (Reference Esser, Bachmann, Kuhn, Schuldt, Forster, Thiel, May, Koch-Nolte, Yanez-Mo, Sanchez-Madrid, Schinkel, Jalkanen, Craig, Bruchhaus and Horstmann2014) suggests the existence of a large number of possible novel host receptors for PfEMP1; however, it is not clear if any of these could sustain an infection at high parasitaemia.

Two recent reports suggest that on the one hand var expression may be under some level of global control (Merrick et al. Reference Merrick, Huttenhower, Buckee, Amambua-Ngwa, Gomez-Escobar, Walther, Conway and Duraisingh2012) and on the other hand, that two other gene families may be able to support sequestration (Niang et al. Reference Niang, Bei, Madnani, Pelly, Dankwa, Kanjee, Gunalan, Amaladoss, Yeo, Bob, Malleret, Duraisingh and Preiser2014; Goel et al. Reference Goel, Palmkvist, Moll, Joannin, Lara, Akhouri, Moradi, Ojemalm, Westman, Angeletti, Kjellin, Lehtio, Blixt, Idestrom, Gahmberg, Storry, Hult, Olsson, von Heijne, Nilsson and Wahlgren2015). PfEMP1 appears also to be partially controlled at the translational level (Brancucci et al. Reference Brancucci, Witmer, Schmid and Voss2014b). This raises the possibility that the parasite could diversify both the type and total amount of variant antigen expressed on the infected erythrocyte surface.

The important question here is whether the level of sequestration supported by STEVOR and RIFINs in the absence of PfEMP1 could support parasite loads associated with severe malaria. Though knockdowns of var have an important effect on the antigenic properties of laboratory isolates (Chan et al. Reference Chan, Howell, Reiling, Ataide, Mackintosh, Fowkes, Petter, Chesson, Langer, Warimwe, Duffy, Rogerson, Bull, Cowman, Marsh and Beeson2012), they may not fully model the behaviour of non-PfEMP1 VSA in vivo. Warimwe et al. (Reference Warimwe, Fegan, Musyoki, Newton, Opiyo, Githinji, Andisi, Menza, Kitsao, Marsh and Bull2012) observed an association between respiratory distress and rosetting in Kenya. Despite a strong overall association between rosetting and the expression of group A var genes, the observed association was independent of group A-like var gene expression or any other subgroup of var genes that could be identified. One possible interpretation of this is that the association between rosetting and respiratory distress was driven by a subset of parasites for which rosetting is mediated through RIFINs or STEVOR. This highlights the need for full sequence information of VSA expressed under different levels of naturally acquired immunity.

Future research on developing interventions based on PfEMP1 needs to continue to refine definitions of different cytoadhesive phenotypes at the molecular level. Various terms such as ‘rosetting’, ‘VSA_SM’, ‘group A PfEMP1’, ‘DC8’, in the same way as clinical definitions such as ‘cerebral malaria’ (Taylor et al. Reference Taylor, Fu, Carr, Whitten, Mueller, Fosiko, Lewallen, Liomba and Molyneux2004), are essential for generating hypotheses, but what they describe are potentially heterogeneous. More studies are still needed to directly link structure, cytoadhesive function and antigenicity with naturally acquired immunity to clearly defined malarial disease.