Plasmodium vivax: Merozoites, invasion of reticulocytes and considerations for malaria vaccine development

of fluomcent lipid analogues in the surface membrane of ad& malt Setitosoma mansoni. Mol. Biochem. Par&of. 53, 233-240 58 Dixon, D.M. and Martin, R.J. (!993) Patch clamp and chloride channels in Ascaris suurn. Parasitology.__ Today 9,341-W 59 Foley, M. et al. (1994) Photoaffinity labellkg of chloroquinebinding proteins in Plasmodium falciparum. I. Binl. Chew. 269, 69556961 60 Harder, A. tpf al. (1988) Influence of praziquantel and Caz’ on

Plasmodium

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the bilayer-isotropic-hexagonal transition of model mtm branes. Mol. Biochm. Pamsitol. 29.554 61 Gully, D.F. el al. (1994) Cloning of an avermectin-sensitive glutamate-gated chloride channel from Camorhnbditis elegams.

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62 McTigue, ma1 drug

M.A. rl nl. (1995) Crystal structure of a schistosoand vaccine target: glutathione-Stransferase from Schistosoma juponica and its complex with the leading antischistosomal drug Praaiquantel. /. Mol. Biol. 246,21-27

vivux:

h&ion of Reticulocytes and for tJ)ti~aria Vaccine Development M.R. Galinski and J.W. Barnwell

Several Plasmodium vivax merozoite proteins have been characterized over the past few years, including two that bind specifically to reticulocytes. Here, Mary Galinski and john Barnwell examine P. vivax meroroites and constituent molecules thnt are involved in host cell selection and invnsion, and that also are viewed as malaria vaccine candidates. They also discuss hotu kwwledge of the reticulocyte-binding proteins furthers the development of a cwceptual fmmeuwrk for malaria merozoite invasion at @e inolecular level, not only for P. vivax, but for all species @fthffhepamite.

PXasmodiumvivnx and P. falciparum are the two most @revalent.species of human malaria, together afflicting a few hundred million people annually’. Although the percentage of these cases caused by P. vivax versus P. falciparum is uncertain, it can be conservati: 1.1~ estimated that as many as 35 million cases \:i vivax malaria occur each year. Moreover, chloroquineresistant strains have recently been observed2-4, and relapse parasitemias due to primaquine-resistant liverstage hypnozoites have become widespread and are being noted more and more frequently5Gh. Vivax malaria is undoubtedly i major public health problem for many countrica, ‘N. h associated socioeconomic ramifications. Now, and probably increasingly so in the future, there is a need for alternative prophylactic and therapeutic tools for control and management of these infections. Despite the fact that P. aivax is a major pathogen, b?sic research on molecular and cellular biological aspects of this parqsite has been limited, with only a f4w laboratories focusing research programs on this $wes. One prominent reason for this lack of attention is that P. vivnx cannov be cultured continuously hi vitra. This contrasts with the availability of a F, filcipurum culture system7 that has greatly facili@ted inves!@tions of i1d.sspecies in many laboratories worldwide. 6hy

k GalhId and )ohn W. Barnwell are at New York University Medical Ce: Iter, Department of Medtcal and Molecu!ar f’arasitology. 34 I East 25th Street NY IO0 IO. USA. Tab + I 212 263 6793H956, Faxz + I 2 I2 263 7475, e-mall: [email protected]

An outstanding trait of P. vivax that has enabled it, to a large degree, to evade scientific scrutiny is that its primary host red blood cell is the reticulocyte, which normally accounts for only about 1% of the human red blood cell population. Reticulocytes are not easily obtained routinely on a daily basis in sufficient quantities to maintain laboratory cultures. This fact also hampers any systematic investigation of optimum culture conditions for P. vivax, which appears to be another important factor affecting the maintenance of this parasite in vitro. Although it is not known why P. vivax is restricted to this young red blood cell population, the discovery of proteins that bind specifically to reticulocytes~ provides the beginning of an explanation for how P. vivax targets thii host cell. This review features the P. vivax reticulocytebinding proteins8 and also outlines work that has been accomplished in recent years to characterize P. vivax merozoites We, and others, have circumvented the fact that there is no culture system for P. vivax by: (1) obtaining limited amounts of this parasite, as required, from Saimiri monkey adapted infections; and (2) by making use of the closely related simian malarias P. cynomolgi and P. knowlesiq, which serve as biological model systems, and are also used to generate homologous DNA ant’ antibody reagents/probes that crossreact with P. vivax. In addition, although there is no long-term culture system for P. vivax, short-term culture with significant reinvasion is possible, thus permitting in vitro investigations on merozoite invasion*OJ1. This body of P. vivax investigations has not only broadened our knowledge of P. vivax merozoite proteins, but also has provided valuable insights about functionally analogous proteins ‘in P. falciparum. vivnr: the parasite The distinct phylogenetic paths of P. viuas and P. falciparum probably evolved tens of millions of years agol2. .This period of time has allowed for considerable biological differences to develop between these predominant species of malaria’““. Most notably: (1) P. vivax can remain dormant in the liver as hypnozoites15, which can initiate a blood-stage infection even months after an initial sporozoite infection; (2) it

Plasmodium

Reviews preferentially, if not exclusively, invades reticulocyteW7; and (3) it develops caveolae-vesicle complexes (Schiiffner’s stippling) in the infected erythrocyte membranel8J9. In contrast: (1) P. falciparum does not develop a dormant liver stage; (2) it invades mature as well as immature red blood cells; (3) it produces knob protrusions in the infected erythrocyte membran@, as opposed to caveolae-vesicle EompIexes; and (4) it ‘sequesters’ its nii~turin& btood-stage fcwms from the circulation &d possible destruction in the s leen by adhering to vascular en %otheIiumz1 (P. vivax do no! seque#er). The dramatic morphological and biological differences between P. vivax and P. faiciparum have indicated that many of the proteins of these two malaria species may be quite different. Despite the distinctive features of P. vivax and P. falcipatum, however, many aspects of the biology of their blood stages are clearly similar. In particular, the merozoites of all malaria species, examined at the ultrastructural level, contain the same features and organelles typical of Apicomplexan parasites: an anterior conoid process (apical cone), paired rhoptries, micronemes and dense bodies (microspheres) (Fig. 1). These structural similarities suggest that, even though each species has different host cell requirements, the general molrcular scheme of events that define invasion Fii. I. Transmission electron micro&# of two f&mod&~ virox merozoites after may be similar across species. Inin viva maturation from trophozoite stage to diirentiated segmented schitonts deed, molecular studies have begun (merozoites) and mechanical release by small bore needle (25G) passage. The organized ‘spiked’ nature of the merozoite surf&e coat (20-25 nm projections) is evident to denote the fine similarities, as in several areas, as noted. Other structures are labeled including the apical cone well as differences, between the (AC), rhoptries (only one of each rhopq pair is visible in each section) (Rh), genes and proteins of these and micronemes (Mn). dense bodies (D) and nucleus (N). Fixation in 2% tannic acid and other species of Plasmodium. Other 2% glutaraldehyde in 0. I M sodium cacodylate buffer. Scale bar = 200 nm. (This image recent reviews and references was kindly produced by Michael J. Stewart-) therein provide accounts of related studies not covered. here, _ in partitilar with regard to the enzymatic and morphoface may participate in and facilitate th: process of logical events known to occur as merozoites enter merozoite entry into red blood cells2’J5. erythrocytesna. The first Plasmodium merozoite surface protein to be identified was the major merozoite surface protein The me&& ‘surface coaY (MSP-1) of P. yoeW6, soon followed by identification ,,Plasmodium vivax mtirozoites have a ‘surface coat’ of the homologous protein in P. fidciparw# and that appears to be composed of a highly structured P. knowlesi”. Merozoite surface protein-l genes have since been character&d in P. falci~rumr)-l(: P. viXSJ2 array of regularly spaced protruding macromolecular complexes (Fig. 1). Although sometimes appearing as and several rodent malaria ~pecies~-~~.It also has ken a ‘fuzzy‘ or amorphous surface, the clear structural shown that MSP-1 of P. fafciplrum, P. Virgo and P. arrangement that we have observed on freshly re knowlesibinds to erythrocytes iu an m vitro eryhoqteleased, rapidly fixed P. vivax merozoites has also been binding assay (EBAYJ’. In recent years, MSP-1 has been observed previously for other species of malaria. evaluated in numerous investigations for its potential Bannister and colleagues have reviewed in detail the as a component of a malaria blood-stage vaccin@. Though structural studies at the resolution of elecultrastructure of the merozoite surface, emphasizing the visual complexity of the surface components tronmicroscopy indicated that the merozoite surface was possibly composed of multiple proteins, other (mostly for P. knuwfesi), and discussing how this surI-

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:,i_ surface proteins were not discovered until more recently. Three P. fulciparum proteins, in addition to PfMsP-1, have been identified that are associated with the merozoite surface and have been named accordingly as MSP-2, MSP3 and MSP-4 (Refs 39,40) (R.L. Coppel, Abstract’); two of these, like MSP-1, are glycosylphosy!&dylinositol (GPI) anchoredJ9 (R.L. Coppel, AbsiiXt’). Siiilarly, in addition to PvMSP-1, we have identified three P. viuux merozoite proteins (PvMSP-2,3 and -4) that appear to be associated with ’ the surface of P. vivzu merozoites (M.R. Galinski and J.W. harnwell, unpublished). It is interesting that, in &#i&p?~ ,’ i.. :““.~,~th’m .oniy &f$P-1 appe$us to be GPI anchored. ~~~~~~. ,. ,.&lick Mdng using sugars !$ t .~~vz&#$~ f&yA acids do not indicate that other major GPI:#+h
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hylo enetically distant malaria species maintain bioP ogica P ly similar proteins, even when divergence may be considerable. Thus, though P. vivax and P. knowlesi merozoites are dependent upon binding to the Duffyglycoprotein, and P. falciparum is dependent upon binding to glycophorin of red blood cells, a distant but nonetheless related molecule performs a similar binding function for each of these malaria species. The red blood cell binding domain of this ‘family’ of proteins has been located within a conserved cysteine-rich motif common to the proteins of all three speciessr~sz. Although many proteins that are localized to the merozoite rhoptries have been characterized and studled from P. &zlci~rurn~, only .one such protein has been characterized in P. vivux. This is the apical membrane antigen-l (AMA-1)54, which is homologous to the relatively well-conserved proteins of the same family that have been described in P. jdciparumjs, P. fragile%, P. chabaudin and P. knowlesis. The precise role this protein plays in invasion is uncertain, although it has been suggested to be a receptor. Apical membrane antigen-l is currently beingevaluated for its potential as a vaccine candidate. There are other rhoptry-associated proteins in P. viuux (J.W. Bamwell, unpublished), but whether some or all will correspond to the rhoptry-proteins characterized from P. fulciparum remains to be determined. Reticulocyte-binding proteins. The P. vivux reticulocyte-binding proteins, PvRBP-1 and PvRBP-2, are high molecular mass molecules that have relative mobilities &I) of about 275-2&0kDa by SDS-PAGE, are predicted to be largely a-helical in nature, colocalize at the apical pole of mero&tes, and bind to reticulocytes in an EBAs. Furthermore, there are two RGD amino acid motifs in PvRBP-I, which potentially could serve an adhesive function of ligand binding on the reticulocytp@. The exact subcellular localization of these proteins remains to be determined and for this reason they have not been categorized above as surface, microneme or rhoptry proteins. However, by immunofluorescence (IFA) they both produce a pattern that is unique when compared to other known proteins viewed in this manner. They do not appear as a ‘double dot’ pattern typical of rhophy localized proteins, nor the single ‘punctate’ apical dot observed for the Duffy-binding protein and sialic acid binding protein, EBA-175, which could be viewed as markers for microneme localization. Both of the PvRBPs give a fluorescent pattern that can be described as an apical crescent or cap that seems to cover the apical pole, extending beyond the conoid process. Each of these large proteins also has a putative transmembrane domain at its C-terminus, which preceeds a short cytoplasmic domain, suggesting that they are membrane-bound and that the ma@ portion of both proteins is externalized. The binding of these proteins to reticulocytes is dramatic”. When reticulocytedepleted human red blood cell populations are used in an EBA there is no binding of the PvRBPs, whereas when reticulocyteenriched red blood cells are used, there is abundant binding. Adhesion of these proteins is independent of the presence or absence of the erythrocyte Duffjr glycoprotein, indicating that they are binding to a novel receptor(s) on the reticulocyte membrane. Furthermore, binding of these proteins to reticulocytes of Parosrtology

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vol. 12. no. I, I996

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usis retiz!ocyte-enriched red blood cells kofnasquirrelmcdcey(padI)andimmunct . . prwpltatknz of culture supematant from ruptured schlzonts that had been metabolitally labeled with Smethkmine (panel 2). usingrabbit amisefum spedfic fw RrRBp-I. Samples were analysd on a 442% @iem SDs-pdyrcrytami gel. M-1 migrates at -28QkDa under redwing (It) condldons, and wer 5OOkDa under nonreducing (NR) condiions. The RWIIRW-2 ‘&&let binds ph&yJ is co-p=ipbfed (panel 2). - remamsasa monomer under NR conditiOme. As previously mported. the Inti-FvRRP-I antibodies as anatysed by western immunoblor. do not crossreact with FvRW2(Ref.&The MOkDabandinpanel I

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l70-180!~Daisagelarcifactcausedbymigtation of a large amount of intact. nonreduced IgC and does not represent a specific imm~noprecipitated malaria protein. Pre-stained protein standards (GibcolSRL) and apoipoprotein 8 (Sigma) were used as molecular markers. Schematic representing a hypothetical model of the PvRBP complex (b). lared on primary amino acid sequence, secondary structure prediction. preliminary localization studies, and biochemical analyses. Both RrRBP-I and PAW-2 polrpeptide chains have putative transmembrane domains and short cytoplasmic tails, and as such are depkted embedded in the outermost mqnbane of the merozoite. The two PVRBP-I monomers are drawn with inua- and incerwhich of the I6 thin disulphide bonds, consistent with experimental data indicating the present e of these covalent bondt; however, &nes (Cs) are actually involved in diiie-bond formation is not yet known. The close proximity and positioning of PVRBP-2 monomers to PVRBP-I indicate that noncovalent interactions may be responsible for their apparent association. The yellow area of PVRBP-2 represents the region of highest homolw with the P. yo&i protein, pY235 (Ref. 69) (see text for details). The RGD amino acid motif’s of PVRBP- I, and C-terminal 4-mer repeased amim acid motifs of M-2 are also indited.

other primate species correlates with their susceptibility to P. vim+. For example, the PvRBPs bind to Saimiri modcey reticulocytes, which are invaded by P. vivax, but do not bind to rhesus monkey reticulocyks, which are not invaded by P. vivux. One of the most striking features about the PvRBPs is that they seem to form a multimeric complex. The most compelling experiments, supporting a multimerit structure, demonstrate that PvRBP-1 migrates as a homodjmer in nonreducing SDS-polyacrylamide gels, while PvRBP-2 migrates as a monomer under these conditions. This is evident in western immuno-

blot analysess, and also when proteins from EBA elutions or immunopreclpitates of Wmethionine labeled merozoite material are analysed by autoradiography (Fig. 2a). Reticulocyte-binding protein-l contains 16 cysteine residues, clustered into four regions, some of which could participate in the formation of interchain, as well as intrachain disulphide bonds. Moreover, both of the PvRBP molecules tend to bind red blood cells in EBA analyses and coprecipitate with antisera specific for one or the other proteins. In light of current data, we propose that PvRBP-1 is a homodimeric membrane-bound protein 23

:;. ‘At & clepend en t on the formation of interchain ’ &&lphide bonds between monomers. Furthermore, since PvRBP-2 often co-precipitates with PvRBP-1, it seems to associate with PvRBP-1 through noncovalent interactions, leading us to terhtively propose a basic model of the PvRBP complex as illustrated in Fig. 2b. ~rtcess

of invasion: unfolding

a molecular

Plasmodiutn invasion of red blood cells entails a complex series of interactions between merozoite and 1. The ad,hesive surface molecuks y niqiy semi! the $nlrpose 6f ‘capturing as tIjey m&b contact in the circulation. k. knovin to be the ‘business end’ where invasion is initiated. Some8Wtimes .@orient&ed such that the apex of its anterior end is juxtaposed directly with the surface of the erythrocyte where formation of a tight junction is initiated. Although microscopic investigations, beginning 25 years ago, elegantly detail the sequence of events that occur as a merozoite attaches to and gains entry into a red blood cell, we are only : beginning to understand the encompassing molecular processes. Especially with the discovery and basic characterization of several merozoite proteins that adhere to red blood cells in EBAs, a framework is emerging upon which we can begin to define the ::; ~+qi&s of molecular events that constitute the early :;I h&s of invasion: merozoite attachment, apical : J&a positioning with the red blood cell surface, and ::: ’ junction formation between the merozoite and the red ;/,.~+&td ctt!l membranes. ’ It is known from videomicroscopy that, prior to spical contact with erythrocytes, merozoites can attach reversibly at any point along their surface. Video,: microscopy and electronmicroscopy studies also suggest that apical reorientation is facilitated by the ,. adhesive merozoite surface coat243. These visual images indicate that the merozoite surface assists in ‘docking’ the merozoite at the surface of a red blood cell, and also aids the merozoite as it comes to be positionally reorientated, as fibrillar extensions between the merozoite surface and red blood cell seem to maintain the necessary associations. In a general scheme for relating adhesive properties of merozoite proteins with function, it is reasonable to suggest that this initial attachment may involve the binding of MSP-136-n, perhaps in conjunction with other known or unknown merozoite surface proteins, and may ;: very well facilitate reorientation. :, : ,: After reorientation, an electron-dense ‘tight junci* tion’ forms between the apical cone of the merozoite i: *and the erythrocyte membrane62,6J. This electron;: dense junction moves posteriorly and circumferen: tiaBy over the merozoite as the erythrocyte mem~;i,brane invaglnates and the merozoite enters the host ; cell. Electronmicrograph analysis of P. knorolesi in: $$on has shown that this junction does not form ~?&%veen P. knuu~lesimerozoites and Duffy-negative ied blood cells+ The interpretation has followed that the binding interaction of the DBP of P. krtorolesi or P. uiva~ with the Duffy glycoprotein either initiates the formation of, or forms a part of, the ‘moving junction’. By inference, the EBA-175 member of this 24

family of adhesive proteins would serve the same function for P. falciparum merozoites. This generalized ‘two step’ scheme for describing the initial contact of merozoites with erythrocytes and junction formation, now can be expanded for P. vivax to include a third component, namely PvRBP binding, to satisfy the requirement of reticulocyte selection. The predicted structure, position on the merozoite and adhesive feature of the PvRBPs together suggest a multifunctional role for these proteins. We have proposed that the PvRBPs target the reticulocytes for invasions. From this perspective, binding of these proteins would commit a merozoite to an appropriately selected host cell. When a vivax merozoite is released into the blood circulation, it is z&rrounded by a preponderance of normocytes, cells it does not invade, but presumably comes in direct contact with. If reversible adhesive contacts imparted by the merozoite surface coat occur as normocytes are encountered (PvMSP-1 binds to normocytes, as well as reticulocytes), committed interactions must not occur until a reticulocyte is detected (Fig. 31. The specificity of the PvRBPs for reticulocytes strongly suggests that they bind to a specific reticulocyte ligandfs). This necessary specific adhesion event could possibly serve the added functions of (11 stabilizing the weaker, reversible contact(s) between the merozoite and host cell that could be directly mediated by MSP-1 and/or other undassified adhesive surface proteins, and (2) enabling the ensuing adhesive reactionfs), leading to junction formation, as well as the invasion process to proceed. It is likely that some signal is needed to trigger the release of the PvDBP, at the appropriate time, from its sequestered placement in the ‘micronem&. A case could be made to support the possibility that MSP-1 initiates such a signaling event once contact occurs between this GPI surface-anchored protein and a ligand on the erythrocyte, as GPI (free and proteinbound) has been reported to activate intracelhrlar signal transduction pathway&*. This could seem plausible for malaria parasites such as P. knowlesi or P. fdciparum that invade normocytes. In this scenario, merozoites would be ‘primed’ to initiate junction formation once the appropriate apical orientation occurs. However, this precise series of events would not seem to be appropriate for a parasite, such as P. aiuax, that is destined to invade reticulocytes. Given the 100-fold greater number of normocytes, premature release of the PvDBP would result in the irreversible abortive binding of the majority of P. uizw merozoites to mature erythrocytes (like PvMSP-1, PvDBP binds to both mature erythrocytes and reticulocytes). In this case, and in the absence of other known recep tors, we propose that PvRBPs, upon binding to their red blood cell receptor(s), are in a position to trigger the release of the PvDBP. This model is compatible with the postulate that the PvRBPs bind and select reticukxytes prior to the progession of other irreversible processes. Activation of the appropriate signaling pathway(s) by the requisite binding of the PvRBPs would ensure that the PvDBP is only released in a timely manner. This scenario needs to be investigated furtherz’br. What form(s) or mechanism(s) of signal transduction might occur remains largely unknown. What is known, however, is that PcmsitofogyTo&y, ~0’01. 12. no :, I996

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staurosporine, an inhibitor of serine/threonine kinases, inhibits entry of P. knowlesi merozoites into red blood cells, but not attachment or junction formationbs. This suggests that a phosphorylation pathmight affect a microway tubule/act&based motor that is involved in merozoite junctional movement and host cell membrane invagination rather than signal transduction events just prior to actual invasion. These mechanisms deserve further investigation, as they hold considerable potential for the development of chemotherapeutic interventions that could target crucial signaling pathways and inhibit parasite multiplication.

merozoites by IFA in a pattern that is identical to that seen on merozoites of P. oizm and P. cynomolgi (G. Rosas-Acosta, M.R. Galinski and J.W. Bamwell, unpublished). Third, EBA analyses of P. faiciparrrm proteins from culture supernatants demonstrate that two high-molecular-mass proteins, reminiscent of the PvRBP high-molecular-weight doublets, bind to erythrocytes (J.W. Bamwell, unpublished). We have tentatively called these the Pi falciparum normocyte-binding proteins (PfNBPsl. The most convincing evidence for the existence of analogous proFig. 3. cartoon illunmi,q the pliit of teins among distantly related the pkamocfii~vi~~~ merozoite. T&n to species of Plasmodium comes from 16 P. vivm merozoites are produced the comparative analysis of the from each schizont. When released PvRBP analogdhomologs structure of a large-moleculariktto the circulating blood, they may If, as postulated, the PvRBP encounter 50-200 normocytic erythmass (235 kDa1 merozoite protein complex serves the multifunctional rocytes before ‘bumping into’ a reticuof P. yoelii tPy2351, which localizes locyce. Logical reasoning would indirole of reticulocyte recognition and to the rhoptries and has a roie in cate that homing co a reticulocyte perhaps signaling for P. uivux the invasion of normocytes@‘. would require a specific mechanism, merozoites, then from an evoluWithin the deduced peptide such as a reticukxyce receptor, ‘in tionary perspective it also is reasequence encoded by the Py235 place’ at the time merozoices are sonable to propose that functiongene, there is a region. (of 500 released from an infected cell. The effially and structurally analogous amino acids) that exhibits a sigciency of P. vimt sekdon and invasion merozoite molecules might be presnificant degree of homology to the Df reticuloqteN7 is attested by the ent in all species of Pfusmodiums. partial sequence reported for fact thas during the acute phase of an Plasmodium cytwmolgi, a reticulomfectkm in either rum-immune humans PvRBP-2 (29.6% identity, 51.6% +-preferring closely related or spknectomized monkeyx P. vivox similarityl@ (Fig. 2b). The signifimultiplie+s by eight- to l2-fold between simian malaria, contains proteins cance of this homology is suptach 48 h cycle of growth. homologous to PvRBP-1 and ported further by our more-recent PvRBP-2. However, concurrent analysis comparing the C-terminal experiments showed that PvRBP gene probes did not regions of both proteins. Approximately 40% of the cross-hybridize to the DNA of P. kndesi, P. fdcipPvRBP-2 sequence (3.75 out of 9.5 kbl had been arum or P. berghei, even under conditions of lowered reported previouslyR. This complete gene has since StringenCye. In addition, antibody crossreactivity was been cloned and analysed (M.R. Galinski, unpubnot observed when initial PvRBP antibody preparlished). Interestingly, there is no additional significant ations, which recognized a limited central portion of sequence homology between this and thr Py235, but each protein, were tested for reactivity with these structurally they appear to be related. Both proteins parasites. However, observations (see below) lend contain aproximately 400 amino acids C-terminal to support to the hypothesis that a molecular complex the previously noted homology, and both have a comparable to the PvRBPs is present in other species putative transmembrane domain that is immediately of Plasrwditim. preceded by a set of short repeated amino acid motifs First, a precedent exists in earlier studies of and followed by an apparent cytoplasmic domain P. knowlesi that would suggest that other proteius (Fig. 2b). Thus, Py235 and PvRBP-2 appear to belong function apically prior to the interaction that leads to to the same protein family. As these two highly junction formationQ. In the absence of the erythrocyte diverged species seem to manifest a gene product Duffy glycoprotein, the merozoite can remain apithat is functionally and structurally similar, it is reatally attached via long fiber-like macromolecules. sonable to assume that a related gene is present in P. What comprises these fibrils? These long-ranging fibfnlcipwum and in other species of malaria. rils would be consistent with the large, hydrophilic Alternative receptors fulfill multiple requirements and a-helical molecular structure, predicted to be characteristic of the PvRBP for RBP-related) macroIt has become evident over the past decade that motecules. Moreover, it is relevant to note that as the P. fufciprunz is capable of utilizing alternative recepjunction between the merozoite and erythrocyte tors to reqnize and gain entry into red blood membranes ‘moves’ around the merozoite as it enters cell+r4. Not all strains of P. falcipnrunz require sialic acid or glycophorins for merozoite invasion and it is the red blood cell, the apical pole remains ‘attached/ anchored’ via an electrondense region25.a that has now believed that at least two merozoite receptors exist that are functional alternatives for EBA-175. Some the appearance of these fibrillar components. Second, strains of P. fnlcipnntm will invade erythrocytes that rabbit antisera recently produced against recombinantly expressed partial segments of selected regions lack the appropriate glycophorin A ligand, but retain glycophorin B, and othel3: invade erythrocytes lacking of PvRBP-1 recognize P. fdci~rum and P. knowlesi Pms;cchgy

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25

* bQth glycophorin A and B ligands; cells lacking glycophorin A do not bind EBA-175 (Refs 51,74). As these alternative merozoite receptors are not yet identified, it is not known whether or not they belong to the same gene family as EBA-175. The H strain of P. knuwhi, on the other hand, is known to contain three genes in its genome that comprise the DBP family (a, J3and 71, all of which are expressed? This ‘redundancy’ provides an explanation as to why either anti-Duffy glycoprotein antibody-mediated blockade or enzymatic removal of the Duffy determinants does not block invasion of P. knowlesi into rhesus monkey erythro-

cells, and likely perform the role of the DBP that is necessary for junction formation in this primate host. In addition, the Py235 gene, which (as noted above) appears to be a homolog of PvRBP-2, is one member of a large gene tamicyx. ‘In fact, multiple bands have been observed when immunoprecipitates of Py235 are electrophoresed on 5% polyacrylamide ge1s’h.n. Plnsmodiutn y&ii is known to comprise subpopulations that can invade normocytes and reticulocytes (lethal parasites) and subpopulations that are restricted to I ~,e$u&ytes (non-lethal parasites). Proteins expressed mnt members 0 the Py235 gene famrly may : 1have differing red blood cell specificities, perhaps i:accoun&rg for the observation that an antiPy235 !,; monoclonal antibody (mAb) passively protects mice 1,‘; ,infected with the virulent YM strain of P. ydii, appar: ently by restricting the parasites to reticulocyte mvasion*. Interestingly, this would suggest that, in P. ye&i, this RBP-Z-like protein binds to normocytes. Moreover, as we do not know SNhetheror not a protein similar to PvRBP-1 exists in P. yoelii, it remains possible that another distinct protein, such as a PvRBP-1 homolog with reticulocyte specificity, is expressed in the non-lethal parasite population and performs the function of reticulocyte selection/adhesion. Are alternative adhesion receptors expressed in P. vivax merozoites? Prior research has indicated that in humans, susceptibility to P. vivnx is absolutely dependent upon expression of the Duffy glycoprotein on erythrocytes79.a. In keeping with this view, a mAb specific for the Duffy glycoproteinR1 blocks P. vianx merozoite invasion of human Duffy-positive reticuloqtes in vitro by 80-90% of the control cellsi”. Howj.. &ver, data now exist that hint at the possibility of alternative receptor molecules with specificities for L : ‘molecules other than the Duffy glycoprotein. First, : :pVDBP does not adhere to the Duffy lycoyrotein of !. : scyuirrel monkey erythrocytes, thoug a the squirrel I ‘monkey is susceptible to P. vivnxll. In addition, the I$u$y glycoprotein mAb only partially blocks invasion of P. v&x into owl monkey reticulocytes, despite ,” the fact that the anti-D&y mAb abolishes binding of the PvDBP to the erythrocytes of this primate*” (J.W. Barnwell, unpublished). These data strongly suggest that P. vivax merozoites can express a protein(s) that does not bind to the Duify glycoprotein, but may perform the function of the DBP. Redundancy in functionatly similar merozoite adhesins 26

could be a ‘fact of life’ in parasitism by Plasmodium species, including P. vivux. We are pursuing investigations of a 145 kDa merozoite protein, named P. viuax merozoite apical cone protein, or PvMacP (M.R. Galinski and J.W. Bamwell, unpublished) that, based on binding and location, could be an alternative receptor to the PvDBP. This protein binds specifically to squirrel-monkey and owl monkey reticulates. On mature segmented schizonts, PvMacP localizes by IFA to the apical pole of merozoites, sometimes as a small fluorescent double dot, but mostly as a single dot. immunoelectronmicroscopy shows that, on free merozoites, PvMacP localizes consistently to the apical conoid process, If PvMacP operates in the invasion of primate host cells as an alternative to the PvDBP, does it also function in merozoite invasion of human red blood cells in vlvax maIaria-endemic regions? The answer to this question will require further investigation of P. vivax isolates acquired directly from humans. However, we have determined that PvMacP was consistently expressed in the majority of merozoites from a sample of 20 p. vivnx isolates taken directly from human infections. For one thing, this suggests that monkey adaptation did not select a minor population of parasites expressing this protein, or induce expression of a silent gene, as has been suggested to occur in P. falcip~un~ during adaptation to an alternative invasion pathwayn, but rather, that PvMacP is another adhesive apical protein that is generally characteristic of P. uivax merozoites. Another question is whether or not there is functional polymorphism in the PvRBPs. Both of the PvRBP genes are present as single-copy genes. Although little is known about the diversity of the PvRBPs, some useful information is available .from studies of P. cynomolgi. Different strains of P. cynomolgi are known to vary in their relative reticulocyte ‘preference’, which, at best (for P. vi>.!i:. or P. cynomolgi), can be defined as restriction of early ring-stage parasites to morphologically identifiable reticulocyte9. We also have found that the restriction enzyme pattern differs for the RBP genes of different strains of P. cynomolgi. Diversity may be one means to provide alternative binding sites. Also, it is conceivable that the PvRBP molecules could associate fdimerize) in different ways to form the RBP complex, thus creating alternative binding domains (or, perhaps, altemative signaling ‘instructions’)83. Also, high parasitemias (much greater than 1%) occur occasionally in human P. vivax infections”4, perhaps indicating that invasion by some strains of P. uivax may not be confined strictly to reticul tes. The notion, that functional polymorphism inT t e RBPs of P. z&ax is plausible, is also supported by the precedent of a multigene family for the RBP-Z-like analog of P. yoelii7h. Plasmodium Vivax merozoite vaccine(s): current considerations Anti-merozoite vaccines can be envisioned to occur by several different antibody-mediated mechanisms, including merozoite agglutination*s, cytophilic antibody dependent toxicityP5, or interference with biological processes such as merozoite protease activity%. The concept of preventing malarial merozoitc invasion into erythrocytes by interfering with critical PLfUSltOtOgy 7cxlu~, vol. I2. no. I. I996

Reviews receptor-ligand interactions through vaccination is alsor!uite attractive. In developing a vaccine of this nature, a number of fvtors must be considered, including: (1) the identiflcation of adhesion domains responsible for ligand binding; (2) determination of their structure; and (3) reproduction of this structure through recombinant expression or synthetic technologies. The goal must be to produce immunogens that will induce antibodies able to recognize the corresponding binding domains of the native merozoite adhesins. Five P. vivnx proteins (PvMSP-1, PvRBP-1, PvRBP-2, PvRBP qnd PvAMA-I) could be viewed today as potepiiai candida@ for develo ent of ti anti-merozbte vaccine that mterfexes wit R” receptor-ligand interactlons. In the case of the PvRBP complex, the reticuloq&-binding domain(s) has not yet been identified, however, and invest@tions towards this end are actively under way. A few major considerations in this search are that the binding domain(s) (1) may be located on either one or both of the RBP molecules, (2) may be conformational, and (3) possibly only form after dimerization of both proteins. The two RGD domains of PvRBP-1 provide one set of clues that could lead to the identification of actual binding regions, although it remains to be determined if these are functional bindin sites. On the other hand, the domain responsible Por the binding of PvDBP has been determinedsz. However, an efficient method of production of this domain, which retains the structural basis for both adhesion and lmmunogenicity, as well as the detailing of the finer structural determinants responsible for the specificity of the adhesion., still needs to be resolved. Further, the structural basis of the adhesion of native (pre-processed) MSP-1 to erythrocyte@J has not yet been thoroughly investigated for P. vivax or P. falcipurum, and the putative rece tor function of the AMA-l protein familyw58 et?! s to be clarified and, hence, possible binding domains identified and evaluated. If our supposition is correct that the PvRBPs target the young red blood cells for invasion, the PvRBPs (and RBP-like analogues) may be one of the critical targets for antibody-mediated neutralization of merozoite invasion. By preventing the ‘commitment’ step of attachment, the initiation of the irreversible steps of PvDBP binding (or, likewise EBA-175 binding) and junction formation would probably be hindered, and invasion should not occur. Furthermore, if transduced signals are needed for release of the PvDBP from micronemes, and if the binding of the PvRBPs is an idtiating component of this signal, then antibody binding to the PvRBPs, theoretically, could induce such signals and cause premature release of the PvDBPs. This would have at least two possible effects. Importantly, the PvDBPs would be much more readily exposed to the action of anti-PvDBP antibodies prior to junction formation. It also is conceivable that this premature signaling action could cause abortive invasion attempts by ‘encouraging’ the irreversible attachment of merozoites to mature erythrocytes. However, in the unlikely event that P. vivax, under such unusual circumstances can invade (and survive in) normocytes, it must also be considered that the release of DBP might lead to an enhancement of infections due to the promotion of junction formation and Parasrcology Today, vol.12.no. I, I996

invasion. An anti-PvRBP vaccine could also, potentially, select for a minor parasite population that can invade normocytes, perhaps making P. vivnx a more virulent parasite. We raise these possibilities (not knowing how likely they may be), in part, to highlight the absolute importance of identifying potential receptors that operate as functional alternatives to primary receptors, whether for P. vivax or P. falciparm. Likewise, it will be necessary to determine the extent and functional significance of polymorphism (structural and functional) in the receptor molecules, and the relationship of this polymorphism to immunogenicity. For example, the adhesion. domain of DBP has been found to exhibit amino acid polymorphilm in human isolate@‘, Will these amino acid changes affect an&d binding and, thus, neutralization of invasion, slmi 49r to that which occurs as a result of variation in the receptors of viruses? In either case, immunization against a major population, potentially, could select minor variants that express alternative receptors or structural polymorphisms, rendering an effective vaccine partially or completely ineffective, while foresight and further basic research may prevent such occurrences. Regardless of the above cortsiderations, the time between merozoite release and completion of host cell entry is short, a matter of a few minutes at most, and the inclusion of several receptor components together in a formulated cocktail merdzoite receptor vaccine cumulatively to hinder successive interactions with host cells will most likely be required to obtain a high level of efficacy. Moreover, the incorporation of other P. vivux merozoite molecules, such as epitopes of PvMSP-3 and -4, which may induce merozoite agglutinating and cytophilic antibodies, may help to achieve a greater maximum and broader protection. Concluding remarks With five P. vivax merozoite vaccine candidate hinding proteins now under investigation, there clearly has been significant progress in the field of P. vizvncvaccine research. In essence, the development of vaccines targeted at P. vivax merozoites, given the current state of knowledge, is no longer far behind similar vaccines for P. falciyarum. In fact, the merozoite receptor antigens that are actively being investigated for P. falciprttra include at this time only three candidates (MSP-1, EBA-175 and AMA-l). With consideration of the cascade of molecular events that seems to occur prior to P. vivax merozoite invasion of reticulocytes tie. RBP binding preceeds DBP binding), however, we certain] would include RBP-like analogs to the list of P. f~Yciprutn candidates. We still have a fair way to go towards understanding P. vivax merozoites and the process of erythrocyte invasion. There are also many unknowns with regard to making a highly effective blood-stage vaccine, even if all the 9est’ components were in hand. Nutwithstanding, there has been considerable movement at many levels on basic research concerning the merozoite and in applied research towards developing effective merozoite vaccines. We have outlined much of the progress of recent years, especially with regard to preliminary research involving the identification and characteri;&on of P. viuax merozoite receptors. These moiecules are potentially strong vaccine candidates 27

and further characte+xtion is clearly required. As the more empirical aspects of vaccine development are carried forward, we emphasize the importance of continued fundamental studies to understand malaria blood stages and to elucidate features of their relationship to their host environment. As we uncover more detail about the malaria parasite and its biological processes, we will be in a considerably stronger position to devise more effective immunogenic compounds for vaccimtto!?.

22

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76 Keen, J. ct II/. (1%)) tdentiftcation of the gene for a Plasmodium yoclii rhoptry protein: multiple copies in the parasite genome. MO/. Rior~/rw. /‘rwrrsi/t~/. 42. 241-24h 77 Ogun. S.A. and Holder, A.A. (1YY.l) f’lnswotliw~r !/w/ii: Brefeldin A-sensitive processing of proteins targeted to the rhoptries. /IX/J. /+m?;ilv/. 79, 270-278 7tl Freeman. R.R.. Trtjdosicwirz, A.J. .md C’ro~s, <;.A.M. ~Ytuu)) Pmtective monoclonal antibodies recognising stagr specific merozoite antigens of a rodent malaria parasite. N~r~rrr,* 2~4, 366-w 7Y Miller, L.H. 1.1 n/. (lY76) The resistance factor to Plnsutorfiur~r vivnx in blacks: The Duffy blood group genotype FyFy. N~u End. I. Med. 295. 302-.3&t 80 S&&r, H.G. I’/ RI. (1978) The Duffy blood group and resistance to P/asmodium viva% in Honduras. /\r~. 1. Trq’. Med. t i!J.s. 27,&l-672 Rl Nichols, M.E. VI d. (1987) A new human Duffy blood gmup specificity deftned by a murine monoclonal antibody. /. Er/). Med. 166,776-785 82 McWilson, W., Skinner, J.C. and &inn, E. (196h) Biology of the simian malarlas of Southeast Asia. I. Host cell preferences of young trophozoites of f&r species of Plnsrmdirtrtt. /. hwusi/td. 52, 14-16 X3 Heldin, C-H. (1YYS) Dimeritation of cell surface receptors in signal transduction. Cc,// Ho, 213-223 84 Witzig, R.S. and Barker, R.tI., Jr (lYY4) Atypical P/oslrlodilrt,r viva% infection in Peru. 7‘nrrtr. K SOL. Trap MCI/. //,I/~~. HR. IYn 85 Boulraroun-Tny~)url, H. c’l (iI. (19%)) Antibodies that protect humans against Plosttrotlirrn~ fdcipnrrort blood stages do not on their own inhibit parasite growth and invasion irr vitro, but act in cooperation with monocytes. / /IS/I. Mol. 172, 1633-1641 86 Blackman, M.J. IV rr/. (1991) Antibodies inhibit the proteasemediated processing of a malaria meroroite surface protein. /. I3/I. Mnf. 1x0, .389-193 87 Tsuboi, T. cl sl. (1994) Natural variation within the principal adhesion domain of the Pfnsarodirrm viztax Duffy binding protein. Ijlfczl. IttrrrrrrIr. 62, 45X1--5%

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