The role of Plasmodium falciparum variant surface antigens in ...

review

Human Vaccines 6:1, 84-89; January 2010; © 2010 Landes Bioscience

The role of Plasmodium falciparum variant surface antigens in protective immunity and vaccine development Lars Hviid Centre for Medical Parasitology at Department of International Health, Immunology, and Microbiology; University of Copenhagen; and Department of Infectious Diseases; Copenhagen University Hospital (Rigshospitalet); Copenhagen, Denmark

Key words: antibodies, immunity, malaria, PfEMP1, vaccines, variant surface antigens Abbreviations: CSA, chondroitin sulfate A; DBL, duffy-binding like; IE, infected erythrocyte; NAI, naturally acquired immunity; PAM, pregnancy-associated malaria; PfEMP1, Plasmodium falciparum erythrocyte membrane protein 1; VSA, variant surface antigen; VSA-PAM, variant surface antigens associated with pregnancy-associated malaria; VSA-SM, variant surface antigens associated with severe malaria; VSA-UM, variant surface antigens associated with uncomplicated malaria

There is substantial immuno-epidemiological evidence that the parasite-encoded, so-called variant surface antigens (VSAs) ,such as PfEMP1 on the surface of infected erythrocytes (IEs) are important—in some cases probably decisive determinants of clinical outcome of P. falciparum malaria. The evidence is increasingly being underpinned by specific molecular understanding of the pathogenic processes involved. Pregnancy-associated malaria (PAM) caused by placenta-sequestering IEs expressing the PfEMP1 variant VAR2CSA is a particularly striking example of this. These findings have raised hopes that development of PfEMP1-based vaccines to protect specifically against severe malaria syndromes—in particular PAM—is feasible. This review summarizes the evidence that VSAs are important targets of NAI, discusses why VSA-based vaccines might be feasible despite the extensive intra- and interclonal variation of VSAs, and how vaccines based on this type of antigens fit into the current global strategy to reduce, eliminate and eventually eradicate the burden of malaria.

Introduction People living in many parts of sub-Saharan Africa are continuously exposed to P. falciparum infection, and the cumulative incidence of parasitemia over just a few months can be close to 100%.1 The acquisition of protective immunity as a result of exposure to natural infection [so-called naturally acquired immunity, (NAI)], means that patent parasitemia, clinical disease, and in particular severe and life-threatening malaria, are markedly concentrated among young children (reviewed in ref. 2). Newborns in highly endemic areas are quite resistant to malaria because they possess antibodies transferred from the mother across the Correspondence to: Lars Hviid; Email: [email protected] Submitted: 01/12/09; Accepted: 07/23/09 Previously published online: www.landesbioscience.com/journals/vaccines/article/9602

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placenta, but their risk of clinical disease gradually increases as passive immunity wanes (reviewed in ref. 3). Young children are therefore at high risk of malaria until they have acquired their own protective immunity, which takes several years even in areas of intense transmission.4 In low-endemicity regions susceptibility often continues into adolescence or even adulthood. Importantly, NAI-dependent protection from malaria-related mortality and severe morbidity is achieved earlier than protection from uncomplicated disease, which in turn precedes control over sub-clinical and asymptomatic parasitemia (which is probably never fully achieved). Taken together, this type of epidemiological evidence suggests that key P. falciparum antigen targets of NAI vary substantially, that some variants are more likely to cause severe disease than others, and that the order in which protective immunity to these variants is acquired is non-random. These inferences fit well with evidence that the parasite-encoded so-called variant surface antigens (VSAs), which are expressed on the surface of infected erythrocytes (IEs), are of decisive importance to P. falciparum malaria pathogenesis and clinical outcome, and are all-important targets of NAI. Although this theoretically points to the usefulness of including VSAs in vaccines against malaria, many scientists consider them impractical candidates, due their notorious variability. The Role of P. falciparum VSAs in Pathogenesis and Naturally Acquired Immunity P. falciparum parasites insert electron-dense knobs in the surface membrane of the erythrocytes they infect.5,6 These knobs are involved in adhesion of late-stage IEs to vascular host receptors7 and contain antigens that undergo clonal antigenic variation in vivo.8,9 Furthermore, IE sequestration in particular tissues and organs has repeatedly been linked to severe clinical syndromes such as cerebral malaria, respiratory distress, and placental malaria.10-12 Finally, antigens in the knobs are selective targets of antibodies,13,14 and immune sera can inhibit receptor-specific adhesion of late-stage IEs in vivo.15 These findings have several

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implications. Firstly, they suggest a link between specific malaria syndromes on the one hand and IE adhesion specificity on the other. Secondly, they indicate that susceptibility to severe P. falciparum malaria syndromes reflects an absence of adequate levels of antibodies with specificity for VSAs mediating IE sequestration at critical sites such as the brain. And finally, they imply that NAI to a large extent depends on acquisition of VSA-specific antibodies. I will deal with these in turn. The link between clinical malaria syndromes and IE adhesion specificities. Several studies have indicated that severe malaria is associated with particular IE adhesion phenotypes. Thus, the ability of IEs to adhere to ICAM-1 has been linked to cerebral malaria in some, but not all studies,16-19 and IE affinity for ICAM-1 has been associated with a PfEMP1 DBL domain.20 IEs obtained from patients with severe disease also more often form rosettes (a number of uninfected erythrocytes surrounding a central IE) in vitro than IEs from patients with uncomplicated disease,21,22 an ability that has been mapped to the DBL-α domain of a number of different PfEMP1 variants.23-25 Individual clones contain several genes encoding PfEMP1 variants implicated in rosetting, and can display a number of rosette sub-types (reviewed in ref. 26). Some of these are likely be more important for the development of severe disease than others, since most studies show a substantial overlap between the rosetting phenotypes of IEs from patients with severe and uncomplicated disease, respectively. Furthermore, the rosetting phenotype has been linked to several types of severity,27-29 suggesting that rosetting might in fact be a marker of distinct adhesion phenotypes involved in unrelated pathogenic mechanisms. The relationship between multi-adhesive IEs and PfEMP1 domains on the one hand, and severe disease on the other points in that direction,28,30,31 and might also explain why it has generally been difficult consistently linking any well-defined malaria syndrome to a particular adhesion specificity in vitro. The strict relationship between pregnancy-associated P. falciparum malaria (PAM) and IE adhesion to chondroitin sulfate A (CSA) is an exception from this rule.32 PAM is characterized by a selective accumulation of IEs in the placenta, where they bind to CSA but not to receptors otherwise very commonly used by IEs from non-pregnant individuals (e.g., CD36). Conversely, biologically significant adhesion to CSA appears to be absent among IEs from non-pregnant individuals.32,33 It is by now well-established that IE adhesion to CSA depends on a particular PfEMP1 variant called VAR2CSA.34-37 VAR2CSA contains several DBL domains with specificity for CSA, suggesting that biologically relevant adhesion to CSA involves a critical combination of multiple domains.38,39 The fact that single domains of PfEMP1 variants expressed by parasites not involved in the pathogenesis of PAM can also show affinity for CSA further illustrates this point, and shows that results regarding receptor specificity of recombinant proteins should be interpreted with caution.40-42 The link between disease susceptibility and lack of VSAspecific antibodies. Clinical disease in semi-immune individuals involves parasites that express VSAs to which the patient has little or no specific antibody.43-46 In contrast, such patients often possess antibodies with specificity for unrelated VSAs. Susceptibility to malaria thus corresponds to gaps in the repertoire of

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VSA-specific antibodies. Again, PAM serves as a useful example of this relationship. At the time of their first pregnancy, women in areas with stable transmission of P. falciparum parasites are generally highly resistant to malaria. Nevertheless, they become highly susceptible to infection with placenta-sequestering parasites upon getting pregnant (reviewed in ref. 47). The peculiar adhesion phenotype of placenta-sequestering IEs (see above) suggests that parasites that have this phenotype do not appear in non-pregnant individuals—that it is incompatible with their survival in a non-pregnant host. Indirect parasitological evidence that this is the case was first provided by a study showing that parasitemia in pregnant women from endemic areas (presumably as a result of PAM) very quickly resolved post partum.48 Perhaps more convincingly, P. falciparum-exposed men, nulligravidae, and children do not possess antibodies that can recognize the VSA expressed by placental IEs (VSA PAM/VAR2CSA),33,35,49,50 again indicating that expression of VSA PAM/VAR2CSA is incompatible with parasite survival unless the host is pregnant. If so, the high PAM susceptibility of primigravidae who were previously resistant to infection is explainable by their lack of protective VSA PAM-specific antibody.51 The link between NAI and acquisition of VSA-specific antibodies. Although a clinical disease episode involves parasites that express a VSA variant to which the patient does not have specific antibody, such an episode generally results in acquisition of an antibody response that is specific for that particular VSA.43,45,46 While it seems plausible that the patient would subsequently be resistant to a parasite expressing the same (or a cross-reactive) VSA, it is hard to demonstrate for obvious reasons. As previously, PAM provides the best evidence currently available. Although pregnant woman are much more at risk of P. falciparum malaria than their non-pregnant peers, this susceptibility is highly parity-dependent (decreases with increasing parity).12 The finding that levels of VSA PAM/VAR2CSA-specific antibodies are also highly dependent on parity (increase with increasing parity) suggest that the two phenomena are related.35,49,50,52 In fact, levels of VSA PAM-specific IgG (importantly, in contrast to levels of IgG with specificity for other VSAs) correlate with protection from the clinical consequences of placental malaria, which are mainly maternal anemia, prematurity and low birth weight of the infant,53,54 strongly pointing to a causal link. Acquisition of protective immunity to PAM is thus dependent on acquisition of antibodies with specificity for VSA PAM/VAR2CSA. The Relationship between Disease Severity and VSA Expression IEs obtained from severe malaria patients tend to express VSAs (called VSA SM) to which most (even quite young) P. falciparumexposed children have substantial antibody levels. In contrast, many fewer such children possess antibodies recognizing VSAs expressed by IEs from children with uncomplicated malaria (VSAUM), and levels are generally lower.55-57 These findings suggest that severe disease is caused by parasites expressing VSA SM with substantial interclonal similarity, and which dominate early in life and among those with little or no pre-existing immunity.

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This would explain why severe malaria mainly occurs early in life and why it appears to require only few disease episodes (probably well under ten) to acquire protection from severe malaria,58,59 despite the assumed vast repertoire of VSAs. Consistent with this hypothesis, VSA SM expression has been related to expression of PfEMP1 variants encoded by the so-called Group A var genes,60 which constitute the structurally most homogeneous var gene sub-family.61,62 But why do such virulent VSAs appear earlier than other, less virulent, variants, and why are they less heterogeneous than other VSAs? The in vivo Order of Appearance of VSA Variants is Non-Random The rate of P. falciparum switching among different antigenic and adhesive phenotypes has been estimated in vitro to be approximately 2% per generation.63 If this rate also applies in vivo, it would seem to ensure that all VSAs are present in the circulation after relatively few cycles, even if all the presumably thousands of erythrocytes invaded by the merozoites emerging from a single infected liver express the same VSA (reviewed in ref. 64). Nevertheless, most parasites in a given infection appear to express the same VSA (or a very few and antigenically related ones),43-46 and changes in the dominant type can be spaced weeks apart.9 Furthermore, the order of appearance of dominant VSAs looks far from random.55-57 This conundrum is most easily resolved by assuming that the “effective in vivo multiplication rate” (EMR: the number of successful descendents/generation) of a given intra-erythrocytic parasite depends on which VSA is expressed on the surface of the erythrocyte it infects. The EMR can safely be assumed to vary among VSAs for a given host at a given time, as well as among hosts for a given VSA, leading to an EMR hierarchy of VSAs. Conceivably, this would lead to rapid dominance of parasites expressing the VSA with the highest EMR value, even in cases where the infection is initially very diverse with respect to VSA expression, and probably often before newly acquired VSAspecific immunity sets in. In fact, there is experimental evidence that such a sorting from initial diversity to focused expression does occur. When immunologically naïve adults were experimentally infected with P. falciparum sporozoites, var gene expression in the first generation of parasites emerging from the liver was very diverse.64 However, within the next couple of parasite multiplication cycles there was a dramatic focusing of var gene transcription on Group A genes.64 When the host responds to the infection (e.g., inflammation, acquired VSA-specific immunity), this hierarchy would be expected to change, leading to a population-level switch from one dominant VSA to another. With time, and in the absence of intervention or re-infection, this scenario would furthermore be expected to lead to a chronic infection characterized by steadily decreasing EMR of the dominant variant, and hence decreasing virulence and decreasing levels of parasitemia. In fact, this is what was seen in patients receiving “malaria therapy” against neurosyphilis.65 Furthermore, it would be expected that chronic infections are initially dominated by parasites expressing one of a relatively small and cross-reactive subset of VSAs (imposed

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mainly by functional constraints for high receptor affinity). Later on, VSA-specific immunity-induced re-ordering of the EMR hierarchy would allow parasites expressing less constrained, and therefore antigenically more diverse, VSAs to dominate. There is only limited, longitudinal evidence of this,8,9 but cross-sectional serological and var gene transcription evidence is certainly consistent with this idea.55-57,66-68 For the sequential change in the EMR hierarchy to operate in the manner outlined above, it must be assumed that the immune system focuses on the dominant VSA, whereas minor sub-populations expressing other VSAs are largely ignored. There is good evidence that this is in fact the case.43,45,46 The chronicity and gradual drop in virulence and parasitemia would similarly seem to require that the level of antibodies with specificity for any given variant declines fairly rapidly as soon as all parasites expressing it are eradicated from the body. Clearly this is difficult to assess, but the fact that levels of VSA PAM-specific IgG decline rapidly after delivery,69 combined with the evidence that parasites expressing VSA PAM cannot survive in a non-pregnant host and therefore disappear shortly after delivery (see above), are at least consistent with this assumption. Importantly, immunological memory persists, as evidenced by the rapid re-acquisition of VSA PAM-specific IgG in a subsequent pregnancy.69,70 VSA-Based Vaccines In the absence of treatment, P. falciparum infection is highly likely to progress to severe and life-threatening disease in a nonimmune patient. Although the associated mortality rate is not known, the limited historic evidence that exists indicates that it may be as high as 25–50%. This is in sharp contrast to the situation in many endemic areas, where acquisition of NAI means that only a small minority of infections become symptomatic, and an even smaller fraction results in severe or fatal disease—the case fatality rate is probably well below 1%.71 Together with the evidence that VSA-specific immunity is a central component of NAI, these figures indicate that VSA-based vaccines could have a very substantial impact on P. falciparum malaria morbidity and mortality. At the same time it must be noted, however, that such successful VSA-based vaccines might have very limited impact on parasite transmission, since they would not necessarily substantially affect the persistence of parasites expressing relatively nonvirulent (and probably very diverse) VSAs. This sets VSA-based vaccines apart from all other types of malaria vaccines, which aim at eradicating infections and/or interrupting transmission, and means that even highly effective VSA-based vaccines cannot be the main tool to eliminate or eradicate malaria. However, such vaccines would be extremely useful complementary tools in any effort to reduce the burden of malaria. They would be likely to have a very major impact on morbidity and mortality among those vaccinated, and it is furthermore likely that this impact would be sustainable for long periods without re-vaccination, because immune responses to critical VSAs will “automatically” be boosted as soon as they wane sufficiently to allow re-emergence of parasites expressing exactly those VSAs. But what are the “critical” VSAs suitable for inclusion in vaccines?


VSAs: PfEMP1 and the “extended family”. The P. falciparum genome contains several multi-gene families that encode proteins present on the IE surface with a known or possible role in antigenic variation.72-76 However, by far the most is known about the var gene family and the PfEMP1 proteins it encodes, and PfEMP1 appears to be the major determinant of the antigenic and adhesive properties of P. falciparum-IEs.77-79 PfEMP1 is consequently the only VSA family currently under consideration in vaccines development. Even within the PfEMP1, only few variants are considered. Among those, by far the leading candidate is VAR2CSA, which is being developed for vaccination against PAM. The prospect of VAR2CSA-based vaccines against pregnancy-associated malaria. PAM is a major cause of maternal and perinatal morbidity and mortality. As outlined above, parasites causing PAM express a particular PfEMP1 variant called VAR2CSA, and acquired immunity to PAM corresponds to acquisition of VAR2CSA-specific antibodies. Although VAR2CSA exhibits interclonal variation, it is much less pronounced than for most other PfEMP1, and it appears to contain surface-exposed antibody epitopes that are shared among most or all VAR2CSA variants.80-83 As it is generally believed that the main protective mechanism of these antibodies is to block the sequestration of IEs in the placenta,52 current efforts towards development of VAR2CSA-based vaccines are concentrated on defining epitopes that are both conserved among clones, and at the same time inhibit IE adhesion to CSA.84,85 Several international research consortia currently undertake collaborative preclinical research towards the goal of developing PAM vaccines based on VAR2CSA. Such vaccines are designed to protect specifically against PAM, and are therefore targeted at malariaexposed young women before their first pregnancy, with the hope that the vaccination will induce a state of immunity that corresponds to that acquired by multigravid women (who are resistant to PAM). The prospect of PfEMP1-based vaccines against severe malaria in childhood. The majority of malaria-related References 1.

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Owusu-Agyei S, Koram KA, Baird JK, Utz GC, Binka FN, Nkrumah FK, et al. Incidence of symptomatic and asymptomatic Plasmodium falciparum infection following curative therapy in adult residents of northern Ghana. Am J Trop Med Hyg 2001; 65:197-203. Hviid L. Naturally acquired immunity to Plasmodium falciparum malaria in Africa. Acta Trop 2005; 95:270-5. Hviid L. The immuno-epidemiology of pregnancyassociated malaria: a variant surface antigen-specific perspective. Parasite Immunol 2004; 26:477-86. Hviid L, Staalsoe T. Malaria immunity in infants: a special case of a general phenomenon? Trends Parasitol 2004; 20:66-72. Trager W, Rudzinska MA, Bradbury PC. The fine structure of Plasmodium falciparum and its host erythrocytes in natural malarial infections in man. Bull World Health Organ 1966; 35:883-5. Miller LH. The ultrastructure of red cells infected by Plasmodium falciparum in man. Trans R Soc Trop Med Hyg 1972; 66:459-62.


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morbidity and mortality occur among children under the age of five years in areas of continuous parasite transmission. There is a very large body of evidence pointing to PfEMP1 as very important targets of NAI that protects against mortality and severe morbidity, but the understanding of which PfEMP1 variants are involved, to which receptors they bind, the identity of the epitopes mediating this binding, their interclonal variability etc., is at present fragmentary. Nevertheless, conserved patterns and structures are emerging at a rapid pace, and will assist in the development of PfEMP1-based vaccines aimed specifically at reducing malaria-related childhood mortality and severe morbidity.86-88 Concluding Remarks P. falciparum malaria continues as an intolerable burden to the welfare and economy of a large proportion of humanity. VSAs— and in particular to PfEMP1—appear to be the main targets of the NAI, which is acquired during childhood, and which largely restricts mortality and morbidity to small children (apart from PAM, which constitutes an exception, but an exception that further illustrates the importance of PfEMP1-specific immunity). Furthermore, mortality and severe morbidity appears to be caused mainly by parasites expressing a subset of VSAs that exhibit limited interclonal diversity, to which protective immunity is rapidly acquired following exposure. It thus appears to be feasible to develop vaccines based on these “critical” VSAs. Such vaccines could potentially have a major impact on the burden of malaria, by preventing parasites from expressing these particular VSAs, and thereby markedly reducing morbidity. Acknowledgements

Most of the views expressed here have been developed as the result of regular discussions with friends and colleagues—not least those at Centre for Medical Parasitology in Copenhagen. My thanks are due to them all, both to those who tend to agree with me and to those who tend not to.

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