Pregnancy-associated malaria: Parasite binding, natural immunity and vaccine development

International Journal for Parasitology 37 (2007) 273–283 www.elsevier.com/locate/ijpara

Invited review

Pregnancy-associated malaria: Parasite binding, natural immunity and vaccine development Benoıˆt Gamain

a,1

, Joseph D. Smith

b,1

, Nicola K. Viebig a, Ju¨rg Gysin c, Artur Scherf

a,*

a

c

Unite´ de Biologie des Interactions Hoˆte-Parasite, Institut Pasteur and CNRS, Paris, France b Seattle Biomedical Research Institute, Seattle, USA Unite´ de Parasitologie Expe´rimentale URA IPP/UNIV-MED, EA3282, IFR48, Faculte´ de Me´decine, Universite´ de la Me´diterrane´e, Marseille, France Received 10 October 2006; received in revised form 21 November 2006; accepted 22 November 2006

Abstract Humans living in areas of high malaria transmission gradually acquire, during the early years of life, protective clinical immunity to Plasmodium falciparum, limiting serious complications of malaria to young children. However, pregnant women become more susceptible to severe P. falciparum infections during their first pregnancy. Pregnancy associated malaria is coupled with massive accumulation of parasitised erythrocytes and monocytes in the placental intervillous blood spaces, contributing to disease and death in pregnant women and developing infants. Indirect evidence suggests that prevention may be possible by vaccinating women of childbearing age before their first pregnancy. This review aims to introduce the reader to the implications of malaria infection during pregnancy and to analyse recent findings towards the identification and characterisation of parasite encoded erythrocyte surface proteins expressed in malariainfected pregnant women that are likely targets of protective immunity and have potential for vaccine development. Ó 2006 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. Keywords: Plasmodium; Placenta; Pregnancy; var; Malaria; CSA

1. Introduction The clinical impact of pregnancy-associated malaria (PAM) was first described 70 years ago and since then has been the subject of more than 2000 scientific papers (for review, see Brabin et al., 2004). Adverse outcomes include low birth weight babies, fetal loss, increased perinatal and maternal mortality, maternal anemia and the risk of hypertension in first-time pregnant mothers (Muehlenbachs et al., 2006). However, until recently it was not clear why high parasitemia occurred in pregnant women who otherwise possess significant clinical immunity to malaria. The basis for the accumulation of parasitised erythrocytes (PEs) in the placenta was unknown until it was shown that PEs from placenta bind to chondroitin sulfate A (CSA) and *

1

Corresponding author. Tel.: +33 1 4568 8616; fax: +33 1 4568 8348. E-mail address: [email protected] (A. Scherf). These authors contributed equally to the work.

not to CD36, a common receptor for PE sequestration in the microvasculature (Fried and Duffy, 1996). This was the first direct evidence that a switch in parasite adhesion phenotype could regulate PE sequestration at different microvascular sites within the body and suggested that the placenta supports the clonal expansion of a unique subset of variants to which immunity has not yet developed. Significantly, after one or two pregnancies transcendent antibodies that recognise placental PEs from different geographic regions develop and correlate with protection from malaria (Duffy and Fried, 2003; Fried et al., 1998; Staalsoe et al., 2004). Fried et al. (1998) were the first to show that antibodies against CSA-binding parasites are associated with maternal malaria resistance after multiple pregnancies and also block CSA-binding of placental isolates from different parts of the world, demonstrating the concept of a transcending immune response to the Plasmodium falciparum CSA ligand. These findings suggest that the surface molecule(s) expressed by placental variants have conserved

0020-7519/$30.00 Ó 2006 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2006.11.011

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antigenic determinants and have spurred efforts to characterise these protective antibodies and to induce them by vaccination. How and when natural PAM immunity develops is incompletely understood but will be important for designing a pregnancy malaria vaccine. 2. Pregnancy-associated malaria and natural protective immunity Women develop increasing resistance to PAM infections over successive pregnancies (Brabin et al., 2004). This pattern of parity-specific resistance has been associated with the acquisition of antibodies to the surface of placental PEs (Beeson et al., 1999; Fried et al., 1998; Maubert et al., 1999; Ricke et al., 2000). Early in first pregnancies, women generally lack antibodies that react with the surface of placental binding PEs, which suggests these express novel surface variants. However, by the second trimester (20 weeks) many primigravid women possess antibodies that react to laboratory-adapted CSA-binding lines, suggesting

they have been exposed to placental adherent PEs (Fig. 1A) (O’Neil-Dunne et al., 2001; Ricke et al., 2000; Staalsoe et al., 2001). Consistent with this interpretation, the blood circulation opens up to the placenta about 10 weeks into pregnancy (Brabin et al., 2004) and biochemical evidence demonstrates that low-sulfated chondroitin sulfate proteoglycans (CSPGs) are present in the placental intervillous blood spaces by the end of the first trimester and can support PEs binding in vitro (Agbor-Enoh et al., 2003). Although it is difficult to directly assess placental sequestration during pregnancy, circulating PEs recovered from second or third trimester women are a mixed population of CSA, and to some extent CD36, binding phenotypes, suggesting that most CSA placental parasites do not undergo a full cycle of replication in the placenta but circulate and sequester during the later developmental stages (Beeson et al., 1999, 2002a; Kamwendo et al., 2002; Ofori et al., 2003). Therefore, a likely sequence of events is that women are susceptible to placental infections early in pregnancy, beginning at approximately 10–12 weeks, and upon

Primigravid

A

Multigravid

Anti A Anti A+B Anti A+B+C+D+E+F

Antibodies

B

Parasitemia

Antibodies

Parasitemia

A

Anti A+B

C

12

24

36

12

Time in Pregnancy (Weeks)

D

E

24 Time in Pregnancy (Weeks)

F

36

B VarA

VarB

VarC

VarA

VarB

VarC

VarD

VarE

VarF

VarD

VarE

VarF

Fig. 1. (A) Hypothetical model for pregnancy-associated malaria (PAM) antibody immunity. Women develop increasing resistance to PAM infections over successive pregnancies. During pregnancy women may experience multiple placental infections, each of which may be comprised of several distinct parasite genotypes. Prior to their first pregnancy, women generally lack antibodies to the placental adhesive type but appear to develop these antibodies soon after their first exposure to placental isolates (detectable after 20 weeks during pregnancy). The level of antibodies may wane between pregnancies but is rapidly boosted in multigravid women (detectable after 12 weeks during pregnancy). (B) The schematic compares six different var2CSA alleles containing both diverse and common epitopes. In model 1, a highly invariant cryptic epitope (position 3) is eventually recognized by antibodies and provides protection to most or all var2CSA alleles. Antibodies may also develop to diverse epitopes but they have no role in protective immunity. In model 2, antibodies mainly target polymorphic epitopes that are only partially shared between different var2CSA alleles. Protective immunity requires a repertoire of antibodies that collectively recognise polymorphism at critical protective epitope(s). For simplicity, this is illustrated as a single location (position 3 in the figure) although is it possible that critical epitopes may be distributed on one or more var2CSA domains.

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this first exposure begin to develop antibodies to placental binding isolates (Fig. 1A). The peak prevalence of peripheral blood parasitemia in pregnant women occurs at the beginning of the second trimester (between 13 and 20 weeks gestation) (Brabin, 1983; Zhou et al., 2002). Whether the early antibody response in first pregnancies is able to control placental infections is unclear but it is not uncommon for primigravid women to have infected placentas at delivery. A few studies have investigated the relationship between P. falciparum infection during the course of pregnancy and placental infection at delivery. Most investigators have found a strong correlation between placental infection at delivery and middle (20–28 weeks) or late (after 28 weeks) blood stage parasitemias during the course of pregnancy (Cottrell et al., 2005; McGready et al., 2004). However, no studies have been performed to characterise possible genotypic variation in parasites infecting women over the course of pregnancy as a consequence of new infections. This would help to determine the amount of antigenic exposure to diverse placental isolates that women may experience during pregnancy and how this relates to the development of protective immunity. At delivery, women can present with one or many different parasite genotypes in infected placentas (Kamwendo et al., 2002; Tuikue Ndam et al., 2005), suggesting that they may be broadly exposed to different placental binding variants over the course of a single pregnancy. Monocytes and macrophages are commonly seen in infected placentas and frequently contain malaria haemozoin pigment (Walter et al., 1982). Free malaria pigment or associated in fibrin deposits are also present in placental histological sections and persist in intervillous spaces after the parasites and monocytes clear. Using these criteria, placental infections have been classified into four different histological grades according to the presence or absence of PEs and malaria pigment in the placental intervillous blood spaces; no infection, acute, chronic or past infection (Bulmer et al., 1993). These categories are believed to reflect a natural progression of infection and immunity. Acute infections are defined by the presence of PEs and minimal pigment in macrophages, but not in fibrin. Chronic infections are characterised by PEs and pigment deposition in monocytes or fibrin, while past infections have pigment deposits, but no PEs. Because of the potential for secondary placental infections with new parasite genotypes, the histological pattern may be mixed. The presence of malaria pigment in monocytes and dense monocytic infiltrates, termed mononuclear intervillositis, have been associated with poor pregnancy outcomes (Ordi et al., 1998; Menendez et al., 2000; Rogerson et al., 2003). Monocytes and other host cells may also be a source of inflammatory cytokines that have been associated with placental infections (for review, see Duffy and Fried, 2005). Thus, monocytes may have a role in both malaria complications and resolution of infection.

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Given that placental infections are frequently accompanied by monocyte infiltrates, the question has arisen whether adhesion blocking or opsonising antibodies are more important for protective immunity. Some studies suggest that anti-adhesion antibodies are critical for protection and that such antibodies only develop after the first pregnancy (Fried et al., 1998). However, other studies have reported that both primigravid and multigravid women have adhesion-blocking antibodies that are effective against CSA-binding laboratory isolates, and that the major difference between primigravid and multigravid women is the timing at which these antibodies are first detectable during pregnancy (20 weeks versus 12 weeks) (Fig. 1A) (Ricke et al., 2000; O’Neil-Dunne et al., 2001; Staalsoe et al., 2001; Beeson et al., 2004). The accelerated kinetics in multigravid women suggest that antibody responses might be rapidly boosted upon re-exposure to placental isolates, and it has been proposed that this may be a factor in improved pregnancy outcomes (O’Neil-Dunne et al., 2001). One variable between studies is whether laboratory-adapted or fresh placental isolates are used to measure the adhesion-blocking response. Since the studies that reported an absence of adhesion-blocking antibodies in primigravid women were performed on fresh placental isolates and most other studies have utilised culture-adapted laboratory isolates, it will be important to determine if laboratory isolates are more susceptible to adhesion-blocking antibodies. This would have direct relevance for developing surrogate assays for immune protection. IgG1 and IgG3 sub-type antibodies are predominant in placental infections (Elliott et al., 2005a), indicating that antibodies may opsonise PEs for interaction with phagocytic cells, as well as to block adhesion. A recent report suggests that CSA-binding PEs evade innate (non-opsonic) phagocytic clearance pathways, contributing to parasite accumulation and recruitment of monocytes in the placenta (Serghides et al., 2006). Taken together, these observations suggest that PAM vaccine development should assess the capacity of vaccine candidates to generate CSA adhesion-blocking activity as well as cytophilic antibodies that could overcome the defect in innate phagocytic clearance by facilitating opsonic phagocytosis. A better understanding of how antibodies mediate protective PAM immunity and the targets of protective immunity will be essential for the design of effective vaccine immunogens. Several recent studies report the binding of non-immune IgM (Creasey et al., 2003; Semblat et al., 2006) or IgM and IgG (Rasti et al., 2006) to the surface of CSA-binding PEs. By using heterologous expression of domains in an African green monkey kidney fibroblast-like cell line (COS-7) or Chinese Hamster ovary K1 cells (CHO-K1), it was shown that these natural antibodies target different domains of var2CSA (Creasey et al., 2003; Semblat et al., 2006). The biological function of this binding remains puzzling and it has been speculated that the binding of IgM/IgG may facilitate placental adhesion or promote immune evasion. However, any potential immune evasive functions are

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difficult to reconcile with studies demonstrating that antibodies to the surface of CSA-binding parasites usually develop during pregnancy and are low or absent in children or men (Fried et al., 1998; Beeson et al., 1999; Maubert et al., 1999; Ricke et al., 2000). This suggests that nonimmune IgG binding is very low compared with specific antibodies that develop during pregnancy. We assume that non-immune IgG/IgM is of low affinity and could be displaced once specific antibodies develop against the CSA-ligand in multigravid women. 3. Parasite ligands mediating adhesion to CSA The observation that maternal antibodies are broadly reactive to geographically diverse placental isolates underpins PAM vaccine efforts but also presents one of the central paradoxes for PAM immunity. Although this finding implies that placental isolates have shared epitopes, the only parasite antigens known to be at the PE surface at the time this observation was made were members of a highly diverse P. falciparum erythrocyte membrane protein 1 (PfEMP1) family encoded by var genes (Baruch et al., 1995; Smith et al., 1995; Su et al., 1995). Each parasite genotype encodes 60 var genes (Gardner et al., 2002), which are expressed in a mutually exclusive fashion at the PE surface (Chen et al., 1998; Scherf et al., 1998). PfEMP-1 proteins possess multiple adhesive modules: the cysteine-rich interdomain regions (CIDRs) and the Duffy binding-like (DBL) domains. On the basis of their sequences, CIDR domains group into three different types (a, b and c) and the DBLs into at least six different types (a, b, c, d, e and x) (Smith et al., 2001). PfEMP1 proteins were previously shown to be responsible for the adhesion of PEs to different host receptors such as class B scavenger receptor CD36 and intercellular cell adhesion molecule-1 (ICAM-1) (Berendt et al., 1989; Ockenhouse et al., 1989). Since placental isolates do not bind CD36, this suggests that the parasite could have switched to a non-CD36-binding PfEMP1 variant to sequester in the placenta. While PfEMP1 proteins encode binding activity, they also differ extensively between parasite isolates. To explain the broad antibody response to geographically diverse placental isolates, two distinct hypothesis were proposed: (i) placental isolates induce new parasite proteins at the PE surface that are relatively invariant between parasite genotypes, or (ii) the PfEMP1 variants mediating placental binding may be unusually conserved between parasite genotypes or contain common epitopes. To differentiate between these possibilities, investigators have attempted to learn more about how PEs bind to CSA and sequester in the placenta. The fortunate discovery that in vitro cultured parasites selected for binding to CSA maintained similar antigenic and binding phenotypes to placental isolates (Robert et al., 1995; Rogerson et al., 1995; Scherf et al., 1998; Buffet et al., 1999; Staalsoe et al., 2001; Salanti et al., 2004) facilitated the identification and characterisation of the parasite CSA ligand.

In order to identify the parasite molecules associated with the CSA adhesion phenotype, laboratories selected CSA-binding PEs in vitro and used different biochemical approaches, such as mAb or proteomic analysis, to assess the PE surface. These strategies have identified several new candidate surface-exposed parasite proteins (Fried et al., 2004; Winter et al., 2005), but none of these have so far been shown to be specific to the CSA-binding phenotype, except for the ring surface proteins (RSP) that are exclusively expressed by the early ring-stage of the parasite (Pouvelle et al., 2000). Although several genes encoding members of the RSP complex have been identified, the protein which mediates adhesion remains elusive (Sterkers et al., submitted for publication). However, significant progress was made using molecular biology approaches in identifying and characterising a unique, unusually conserved variant of the PfEMP1 protein family with a role in pregnancy malaria. 4. Var1CSA and varCS2: two puzzling CSA binding var genes In 1999, two var genes (FCR3varCSA and varCS2) were identified as being implicated in the CSA adhesion phenotype of the IT/FCR3 strain by two different groups (Buffet et al., 1999; Reeder et al., 1999). FCR3varCSA, also renamed var1CSA, encodes for a PfEMP1 protein that possesses eight receptor-like domains (seven DBL domains and one CIDR) (Fig. 2), whereas the varCS2 gene encodes for five domains (three DBL domains and two CIDR). By expressing recombinant proteins, it was shown that a DBLc type domain in both var1CSA and varCS2 bound CSA (Buffet et al., 1999; Reeder et al., 1999), but unlike most PfEMP1 proteins, the CIDR domains did not bind CD36 (Gamain et al., 2001, 2002). Structure–function analyses identified a minimal binding region of 67-amino acids within var1CSA DBL3c (Gamain et al., 2004). Although var1CSA and varCS2 had adhesion phenotypes consistent with a role for both proteins in placental binding, they share few structure and sequence homologies. In addition, while var1CSA is relatively conserved in all parasites so far analysed (Rowe et al., 2002; Salanti et al., 2002; Vazquez-Macias et al., 2002; Kyes et al., 2003), varCS2 is not (Duffy et al., 2006b). New transcription studies on CSA-binding parasites showed that varCS2 was a minor variant expressed in the original CSA-binding line from which it was characterised (Duffy et al., 2005) and that var1CSA had an unusual stage-specific transcription and did not appear to undergo antigenic variation (Kyes et al., 2003). These results raised doubts about the role of varCS2 and var1CSA in placental binding. In an attempt to validate the var1CSA gene as ‘‘the’’ CSA-binding ligand, gene disruption studies were performed. Var1CSA disruption mutants were initially unable to adhere to CSA; however, they could recover the phenotype after repeated selection over CSA (Andrews et al., 2003). This study also showed that recovery of CSA-binding was var1CSA-inde-

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Fig. 2. Model showing observed inter- and intra-molecular cross-reactivity epitopes between domains of var1CSA and var2CSA molecules. CIDR, cysteine-rich interdomain region; DBL, duffy-binding like; CSA, chondroitin sulfate A; TM, transmembrane; ATS, acidic terminal segment.

pendent and mediated by another high molecular weight variant (termed var2CSA). Importantly, mAb raised against DBL-c of the var1CSA gene product recognised the var2CSA PfEMP1 (Andrews et al., 2003), indicating the presence of shared epitopes between var1CSA DBL-c and var2CSA (Fig. 2). Var1CSA transcriptional regulation is not typical of other var genes and a definitive function for the var1CSA protein during parasite asexual blood stage development has not been established. 5. Var2CSA: a predominant var gene expressed in placental isolates At the time that researchers were trying to identify the var gene(s) involved in CSA adhesion, RT-PCR experiments were designed using degenerate primers based on the known DBL-a sequences present in the database (Peterson et al., 1995; Taylor et al., 2000). These primers were thought to cover most var genes, but with the release of the P. falciparum isolate 3D7 genome sequence, atypical var genes without DBL-a, or with highly divergent DBL-a, were identified. New primers specific for each var genes present in the 3D7 genome were then designed and used to analyse the transcription pattern of var genes in unselected and CSA-selected NF54/3D7 parasite populations (Salanti et al., 2003). This study showed that only one var gene (var2CSA, PFL0030c) is transcriptionally upregulated in the NF54 parasite lines selected to bind CSA. In addition, a var2CSA ortholog was upregulated in a distinct parasite isolate selected to bind CSA (Salanti et al., 2003). Since the initial discovery, var2CSA homologues have been consistently shown to be upregulated in different parasite genotypes selected to bind CSA, including the var1CSA-disruption mutant (Elliott et al., 2005b; Gamain et al., 2005). Strikingly, var2CSA is structurally unique from all other var genes in the parasite genome. Var2CSA has new types of DBL domains, termed DBLX, and lacks a CIDR domain. Furthermore, var2CSA does not contain a DBLc domain, but instead contains multiple distinct CSA-binding domains (DBL2-X, DBL3-X, DBL5e and DBL6e,

Fig. 2) (Gamain et al., 2005; Avril et al., 2006) suggesting that multivalency may be important for placental sequestration. Although there is limited overlap of variant antigen repertoires between isolates, var2CSA is an exception. Most or all parasite isolates contain a var2CSA ortholog conserved at 75% amino acid identity across worldwide parasite isolates (Trimnell et al., 2006). Curiously, gene orthologs for both var1CSA and var2CSA are also present in the chimpanzee malaria Plasmodium reichenowi, which is believed to have diverged from P. falciparum 6– 10 million years ago (Ayala et al., 1999), indicating that both genes have an extremely ancient origin. It is surprising to have isolate-transcendent members in the var gene family, since most var genes are not conserved across parasite isolates as gene orthologs. This suggests that both var1CSA and var2CSA are under unusual selection to be maintained in the parasite population, potentially at other stages of the parasite life-cycle since both genes could be disrupted in blood stage cultures. To investigate whether other var genes, besides var2CSA, are involved in CSA-binding, the var2CSA gene has been disrupted in two parasite genotypes (Viebig et al., 2005; Duffy et al., 2006b). In both studies, disruption of var2CSA led to a loss of the CSA-binding phenotype but did not affect the ability of the mutants to switch expression to another var gene and display the CD36 adhesive phenotype. Even after multiple rounds of selection, FCR3Dvar2CSA mutant clones did not recover the CSAbinding phenotype (Viebig et al., 2005) or switched to low affinity CSA binders that no longer reacted in a gender-specific manner with multigravid sera (Duffy et al., 2006b). Therefore, it was concluded that var2CSA is the predominant var gene involved in CSA binding. Consistent with this interpretation, var2CSA transcription is highly upregulated in parasites from infected placentas (Tuikue Ndam et al., 2005; Duffy et al., 2006a) and pregnant women exposed to malaria infection acquire antibodies to recombinant var2CSA proteins suggesting this protein may be an important target of protective immunity (Salanti et al., 2004; Tuikue Ndam et al., 2005).

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CSA-binding parasites not only express a particular surface ligand encoded by the var2CSA gene but display changes in the transcription profile of a number of genes compared with parasites expressing CD36 ligands. Most transcriptional changes are observed in genes involved in the modulation of the infected erythrocyte membrane such as the mature parasite-infected erythrocyte surface antigen (MESA) (downregulation) and members of the FIKK kinase gene family (up- and downregulation) (Ralph et al., 2005; Nunes et al., 2007). These changes may be even more pronounced in clinical isolates that have never been cultivated (Daily et al., 2005). Thus, it remains to be shown but it is possible that placental CSA-binding parasites coexpress particular surface antigens together with var2CSA. 6. Antigenic polymorphism in var2CSA A central issue in pregnancy malaria vaccine development is to determine the specificity and antigenic diversity of epitopes targeted by protective maternal antibodies. While maternal antibodies are broadly reactive to placental isolates, it is not clear if placental isolates have highly conserved epitopes or if this cross-reactivity reflects different antibody specificities in the pool of antiserum. Indeed, recent serological comparisons suggest there is considerable antigenic polymorphism in placental isolates which may correspond to sequence polymorphism in var2CSA (Tuikue Ndam et al., 2004; Beeson et al., 2006). To explain how maternal antibodies are able to cross-react on CSAbinding lines and placental isolates from diverse geographic regions, it has been postulated that protective immunity might be mediated by an isolate-transcendent antibody response to highly conserved epitope(s) on var2CSA or another parasite protein, and that once these antibodies develop women are highly protected against most placental variants (model 1, Fig. 1B). According to this hypothesis, the conserved epitopes are likely to be less immunodominant since this would explain the delay in maternal immunity and perhaps why multigravid women remain partially susceptible to low level placental infections. An alternative model is that protective immunity is mediated by a repertoire of antibodies targeting epitopes that are only partially shared between different var2CSA alleles (model 2, Fig. 1B). This hypothesis is supported by recent serological analyses that suggest that var2CSA contains cross-reactive antibody epitopes (Elliott et al., 2005b) and sequence comparisons which show that var2CSA sequences have extensive gene mosaicism leading to a globally related pool of var2CSA sequence polymorphism (Kraemer and Smith, 2006; Trimnell et al., 2006). Under this scenario, the breadth of the maternal antibody response may be expanded during pregnancy by exposure to multiple different parasite genotypes expressing distinct var2CSA alleles. Consequently, PAM immunity may be mediated by a repertoire of antibodies that collectively recognise polymorphism in critical protective epitope(s) that may be on one or more var2CSA domains. This model implies that

PAM immunity may be delayed in endemic regions where women experience fewer multi-genotypic infections. The questions become, ‘‘How many different var2CSA variants do women need to experience to acquire significant PAM immunity, which are the critical epitopes of protection, and how many different antibody specificities are required for protection?’’ The distinction between these two models (Fig. 1B) has implications for pregnancy malaria vaccine design. If protection is mediated through lesser immunodominant, but highly conserved, epitopes it will be necessary to first identify these determinants and then develop vaccine strategies that promote their recognition by antibodies. The specific approach will depend on whether the key protective epitopes reside on var2CSA or less polymorphic parasite proteins at the PE surface that remain to be discovered. Alternatively, if protective immunity is mediated through a repertoire of antibodies targeting divergent protective epitopes that are only partly shared between different var2CSA alleles, then mixtures of different var2CSA forms will likely be required to stimulate an effective antibody response. This may also require targeting of specific regions of var2CSA, such as adhesion-blocking epitopes. Further studies to elucidate the fine specificity of protective antibodies will be critical for designing an effective PAM vaccine. 7. Intervention strategies against the CSA binding ligands A promising avenue for new intervention strategies against PAM is to provide young women with the necessary immunity that would protect first time pregnant mothers and their fetuses against severe complications of placental infections. Either therapeutic antibodies or vaccination using recombinant var2CSA proteins are being considered. Before therapeutic antibodies can be provided to pregnant women, the potential risks to the fetus and mother, dosing schedule and approaches for monitoring the efficacy would need to be determined. Moreover, if the therapy is to take the form of engineered antibodies, then it is still necessary to identify broadly reactive human or mouse mAbs that react with diverse placental isolates. Even in the event that such antibodies cannot be adapted for therapeutic purposes, they may still be valuable for guiding vaccine design, which is considered the definitive solution for pregnancy malaria. Current vaccination strategies are focused on either var1CSA or var2CSA recombinant antigens. Although var1CSA is not considered to have a role in placental sequestration, the recombinant DBL3c domain binds CSA and both baculovirus-expressed and refolded bacterial DBL3c domains elicit antibodies to CSA-binding isolates that block PE adhesion to CSA (Lekana Douki et al., 2002; Costa et al., 2003; Bir et al., 2006). Furthermore, antibodies raised against a refolded bacterial DBL3c domain cross-reacted with the CSA-binding DBL3X domain of var2CSA (Fig. 2) (Bir et al., 2006). Importantly,

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sera from endemic areas recognise the refolded bacterial DBL3c domain and block its binding to CSA in a genderand parity-dependent manner, suggesting that it contains epitopes recognised by naturally acquired protective antibodies from multigravid women. Since there is no evidence that var1CSA is expressed at the PE surface this may provide insight into cross-reacting epitopes between different CSA-binding DBL domains (Fig. 2). It is tempting to speculate that cross-reacting epitopes are the targets of protective antibodies. Given the observed polymorphism in var2CSA, it is likely that the protective immune response against placental isolates needs to include antibodies against different allelic forms. Further studies are necessary to exploit the potential of var1CSA DBL3c for intervention strategies against PAM. More recently, the evidence that var2CSA binds CSA and is upregulated in placental isolates has focused attention on this protein. To date, baculovirus expressed var2CSA recombinant proteins have been able to induce cross-reactive antibodies to at least two different CSA-binding parasite lines (Salanti et al., 2004), although it was not reported if these were able to block PE binding (Barfod et al., 2006; Salanti et al., 2004). In another approach to investigate the antigenic surface of CSA-binding lines, Gysin and colleagues developed a method to tolerise neonatal mice with human erythrocytes prior to immunisation with P. falciparum-infected PEs (Lekana Douki et al., 2002). A large panel of surface-reactive mouse mAbs was isolated that specifically reacted with parasites expressing the CSA phenotype but not the CD36 or ICAM-1 phenotypes. A minority of them, however, showed significant inhibition of cytoadhesion to CSA (20–60% at 5 lg/ml). Surprisingly, all inhibitory antibodies were IgM (Avril et al., 2006). Mapping of the recognition sites of inhibitory mouse mAbs revealed surprising results. All inhibitory mAbs reacted with two or three different DBL domains expressed on the surface of CHO cells and all recognised domains have been shown to be able to mediate binding to CSA (Fig. 2). Thus, it is tempting to speculate that only mAbs that recognise cross-reacting epitopes on multiple CSA-binding domains are potent inhibitors of cytoadhesion (Avril et al., 2006). However, this interpretation is still not easy to reconcile with the sequence diversity in var2CSA and the different var2CSA domains, emphasising the importance of mapping adhesion-blocking epitopes. Efforts are also underway to develop human mAbs from pregnant women exposed to malaria. Interestingly, a number of human mAbs with adhesion-blocking activity were of IgM type (Gysin et al., unpublished observations). Human mAb tools will be extremely valuable to explore the specificity and diversity of epitopes targeted by maternal antibodies.

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syncytiotrophoblast layer or within the intervillous blood spaces where the majority of infected erythrocytes accumulate (Muthusamy et al., 2004). Two host receptors other than CSA have been postulated to play a role in placental sequestration: hyaluronic acid (HA) (Beeson et al., 2000) and neonatal Fc receptors via non-immune immunoglobulins (Flick et al., 2001). The exact role of HA and neonatal Fc receptors in PE cytoadhesion is, however, controversial. Whereas some investigators have reported that specific binding to HA can occur in the absence of CSA binding (Beeson et al., 2000, 2002b; Chai et al., 2001; Beeson and Brown, 2004) and that placental isolates bind to multiple receptors including HA, IgG/IgM and CSA (Rasti et al., 2006), other investigators have failed to confirm the specificity of PE cytoadhesion to HA and concluded that binding of PEs to bovine vitreous humor HA is mediated by CSA contamination in the HA preparation (Fried et al., 2000, 2006; Valiyaveettil et al., 2001). One potential variable is that different approaches were used to assess binding in the different studies. To eliminate this source of discrepancy it would be valuable to introduce standardised binding approaches to compare different studies. More work also needs to be done to establish whether HA and neonatal Fc receptors are accessible to PEs in the placenta. Although earlier studies reported that HA may constitute up to 1–2% of the total glycosaminoglycans (GAG) content in the placenta (Achur et al., 2000; Valiyaveettil et al., 2001), HA is not detectable either in the intervillous space or on the syncytiotrophoblast lining of placentas of various gestational stages between 21 weeks and term, and may have resulted in the total GAG content from umbilical cord contamination in which HA is abundant (Fried et al., 2006). The binding of non-immune immunoglobulins to the PE surface was reported earlier to mediate interaction of PEs to the syncytiotrophoblasts via neonatal Fc receptors (Flick et al., 2001). However, some studies have concluded that neonatal Fc receptors are not expressed by the syncytiotrophoblasts and therefore would not be accessible to PE cytoadhesion (Kristoffersen, 1996; Kristoffersen and Matre, 1996; Lyden et al., 2001). It remains to be seen whether parasite adhesion to syncytiotrophoblasts may induce changes in the placenta leading to the sporadic expression of Fc and HA receptors. Obviously, more work needs to be done to characterise non-CSA placental receptors and potential parasite ligands, and also to determine their respective contributions during PAM. Var2CSA-deficient mutant parasites are useful tools to identify additional host receptors on the syncytiotrophoblasts or in the placental intervillous space.

8. Other receptors involved in PAM 9. Conclusion While CSA is considered the major receptor for placental sequestration, the question arises whether other host receptors are responsible for PE binding to the

Placental malaria is a classic example of malaria research that demonstrates the extraordinary benefit of

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bench work combined with field studies. The initial observation that laboratory parasites can bind to CSA helped frame the unique character of parasites isolated from the placenta. This observation has opened new avenues in the field of PAM and work on laboratory strains has been valuable in identifying the var gene that plays a key role in PAM. Conceptually, we are now moving closer to the development of a vaccine that could protect pregnant women against PAM. However a number of important issues still need to be addressed to better understand the mechanisms of PAM immunity: (i) Do both opsonising and adhesion-blocking antibodies contribute to PAM immunity? (ii) Does var2CSA contain common conserved antibody epitopes or is protective immunity related to the accumulation of antibodies against polymorphic epitopes that are partially shared between different var2CSA sequences? (iii) Are there limits on var2CSA sequence polymorphism and will the protein diversity increase if put under vaccine selection in primigravid women? (iv) Do parasite proteins, besides var2CSA, contribute to protective epitopes? In the absence of an animal model for P. falciparum PAM infections to evaluate these parameters, the development of a PAM vaccine will depend on the quality of in vitro assays that can assess the efficacy of vaccine candidates. It will be important for the PAM scientific community to collaborate in defining standard assays to compare immune responses between recombinant candidate molecules. Acknowledgements The authors are supported by a grant from the Bill & Melinda Gates Foundation (Grant No. 29202) as part of the Pregnancy Malaria vaccine consortium; A.S. and J.G. are supported by the BIOMALPAR program (LSHP-CT2004-503578), an FP6-funded network of excellence; B.G. and J.G. are supported by a grant from the European Malaria Vaccine Initiative (Grant No. 01/2005); A.S. and J.G. are supported by a grant from the ‘‘Fonds de´die´: Combattre les Maladies parasitaires’’ Sanofi Aventis – Ministe`re de la Recherche. References Achur, R.N., Valiyaveettil, M., Alkhalil, A., Ockenhouse, C.F., Gowda, D.C., 2000. Characterization of proteoglycans of human placenta and identification of unique chondroitin sulfate proteoglycans of the intervillous spaces that mediate the adherence of Plasmodium falciparum-infected erythrocytes to the placenta. J. Biol. Chem. 275, 40344–40356. Agbor-Enoh, S.T., Achur, R.N., Valiyaveettil, M., Leke, R., Taylor, D.W., Gowda, D.C., 2003. Chondroitin sulfate proteoglycan expression and binding of Plasmodium falciparum-infected erythro-

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