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 chloroquine- binding proteins in Plasmodium falciparum. I. Binl. Chew. 269, 69556961

60 Harder, A. tpf al. (1988) Influence of praziquantel and Caz’ on

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. N&w 371, 707-711

62 McTigue, M.A. rl nl. (1995) Crystal structure of a schistoso- ma1 drug and vaccine target: glutathione-Stransferase from Schistosoma juponica and its complex with the leading antischistosomal drug Praaiquantel. /. Mol. Biol. 246,21-27

Plasmodium vivux: “~&w&es, h&ion of Reticulocytes and

I &Miwations for tJ)ti~aria Vaccine Development 1; , 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 con- stituent 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 @fthffhe pamite.

PXasmodium vivnx 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 ver- sus 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, chloroquine- resistant strains have recently been observed2-4, and relapse parasitemias due to primaquine-resistant liver- stage 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 atten- tion 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.s species 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: bhy.Galhsld@ccmail.med.nyu.edu

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 quan- tities 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 spe- cifically to reticulocytes~ provides the beginning of an explanation for how P. vivax targets thii host cell.

This review features the P. vivax reticulocyte- binding 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 gener- ate 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 pos- sible, thus permitting in vitro investigations on mero- zoite 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.

Plasmodium 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 con- siderable biological differences to develop between these predominant species of malaria’““. Most notably: (1) P. vivax can remain dormant in the liver as hypno- zoites15, which can initiate a blood-stage infection even months after an initial sporozoite infection; (2) it

Reviews

preferentially, if not exclusively, invades reticulocyteW7; and (3) it develops caveolae-vesicle com- plexes (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 pro- duces 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 destruc- tion in the s vascular

leen by adhering to en otheIiumz1 (P. vivax %

do no! seque#er). The dramatic morphological and biological differ- ences between P. vivax and P. faicip- arum 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, how- ever, many aspects of the biology of their blood stages are clearly simi- lar. In particular, the merozoites of all malaria species, examined at the ultrastructural level, contain the same features and organelles typi- cal 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 re- quirements, the general molrcular scheme of events that define invasion may be similar across species. In- deed, molecular studies have begun to denote the fine similarities, as well as differences, between the genes and proteins of these and other species of Plasmodium. Other recent reviews and references therein provide accounts of related studies not covered here, in par- . _

Fii. I. Transmission electron micro&# of two f&mod&~ virox merozoites after in viva maturation from trophozoite stage to diirentiated segmented schitonts (merozoites) and mechanical release by small bore needle (25G) passage. The organ- ized ‘spiked’ nature of the merozoite surf&e coat (20-25 nm projections) is evident in several areas, as noted. Other structures are labeled including the apical cone (AC), rhoptries (only one of each rhopq pair is visible in each section) (Rh), micronemes (Mn). dense bodies (D) and nucleus (N). Fixation in 2% tannic acid and 2% glutaraldehyde in 0. I M sodium cacodylate buffer. Scale bar = 200 nm. (This image was kindly produced by Michael J. Stewart-)

I -

titilar with regard to the enzymatic and morpho- logical events known to occur as merozoites enter erythrocytesna.

The me&& ‘surface coaY ,,Plasmodium vivax mtirozoites have a ‘surface coat’

that appears to be composed of a highly structured array of regularly spaced protruding macromolecular complexes (Fig. 1). Although sometimes appearing as a ‘fuzzy‘ or amorphous surface, the clear structural arrangement that we have observed on freshly re leased, rapidly fixed P. vivax merozoites has also been observed previously for other species of malaria. Bannister and colleagues have reviewed in detail the ultrastructure of the merozoite surface, emphasizing the visual complexity of the surface components (mostly for P. knuwfesi), and discussing how this sur- Parasrtology T&y, vd. 12. no. I. ! 996

face may participate in and facilitate th: process of merozoite entry into red blood cells2’J5.

The first Plasmodium merozoite surface protein to be identified was the major merozoite surface protein (MSP-1) of P. yoeW6, soon followed by identification of the homologous protein in P. fidciparw# and P. knowlesi”. Merozoite surface protein-l genes have since been character&d in P. falci~rumr)-l(: P. viXSJ2 and several rodent malaria ~pecies~-~~. It also has ken shown that MSP-1 of P. fafciplrum, P. Virgo and P. knowlesi binds to erythrocytes iu an m vitro eryhoqte- binding assay (EBAYJ’. In recent years, MSP-1 has been evaluated in numerous investigations for its potential as a component of a malaria blood-stage vaccin@.

Though structural studies at the resolution of elec- tronmicroscopy indicated that the merozoite surface was possibly composed of multiple proteins, other

21

seen which, if any, surface proteins other than MSP-1 $ :, @d to erythrocytes, as the EBA used to detect the :

-!’ ” .binding of this protein and others may not identify all !i proteins with initial adhesive functions. The nature

id treatment of each culture supematant, as well as the specific binding conditions utilized, affect the out- come of each EBA. it is also possible that other sur- face adhesins exist that bind after invasion begins, once appropriate binding sites become exposed, as has been proposed for the epidermal growth factor (EGR-like domains of the proteolytic processed 19 kDa fragment of MSP-1 (Ref. 42).

The merozoite anterior (apical) pole Microneme and rhopty protein:. The first Plnsmodium

molecule found to be located in the microneme organelles of the merozoite was the 135 kDa P. knowlcsi D&y-binding protein (PkDBP)J’, which is known to bind to the erytluocyte membrane glycoprotein bear- ing the Duffy blood group determinant+45. The gene encoding this protein was subsequently used as a probe to clone the corresponding gene from P. vivux, PvDBp, which encodes a related 14OkDa P. viunx menozdite protein that also binds the Duffy glyco- protein of human red blood cellslr. These two genes were found to be related to the P. falciparum gene encodin the merozoite protein, known as erythro- cJwb4g ti m m an en-175 (EBA-175)a7,@, which was also found to be 7 ocalized to the micronemes49, but bound to sialic acid residues specifically on erythro- cyte glycophorin Am31 and not the Duffy glyco- protein. The relatedness of these P. uivax, P. knowlesi and P. fctlciparum proteins, which had been viewed as unique, species-specific molecules with different host- cell binding specificities, underscores the fact that

- --.---- - --.- ~-.-____ __-. l Vlltth lntematlonal Congress of Parmtol~gy. Izmr. Turkey. Abstwt R21.2(&21) 1994

22

I ',, :,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?~ .oniy &f$P-1 appe$us to be GPI anchored. ~~~~~~. ,. ,.&lick Mdng using sugars

,’ i.. :““.~,~th’m

!$ t .~~vz&#$~ f&y acids do not indicate that other major GPI- :#+h<u,. ~*p..~.~. A &ii:&w@$tiace proteins exist in P. vir%r merozoites. @ ~1~~~~ $&&‘r#tn MSP-2 and lv6P-4 appear to be &$t&@&&&-&&&~~2, -3 and 4. H@weva, fe&ura

of PfMSP-3 (Ref. 41), also known as SPAM, suggest ‘, that there may be a small structural region analogous

to a larger structural domain found in PvMSP-3 and j PVMSPJI.

Although several discrete proteins are now known to be arranged at the surface of the merozoite, it is not known in which way(s) they interact in order to make up the organized array of complexes noted to be pres- ent in ultrastructural studies. The functional role of these proteins is largely unknown. It remains to be

P P hylo enetically distant malaria species maintain bio- ogica ly similar proteins, even when divergence may

be considerable. Thus, though P. vivax and P. knowlesi merozoites are dependent upon binding to the Duffy- glycoprotein, and P. falciparum is dependent upon bind- ing to glycophorin of red blood cells, a distant but nonetheless related molecule performs a similar bind- ing 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 homolo- gous to the relatively well-conserved proteins of the same family that have been described in P. jdcip- arumjs, 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 reticulo- cyte-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, co- localize 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 poten- tially could serve an adhesive function of ligand bind- ing on the reticulocytp@. The exact subcellular local- ization of these proteins remains to be determined and for this reason they have not been categorized above as surface, microneme or rhoptry proteins. How- ever, by immunofluorescence (IFA) they both pro- duce 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 de- scribed 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 puta- tive 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 reticulocyte- enriched 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. Fur- thermore, binding of these proteins to reticulocytes of

Parosrtology ~odoy vol. 12. no. I, I996

Reviews

PvlyP-1

P. wii mwin

4 2 Wrocyte-bindivi -Y (4 perfwmed usis retiz!ocyte-enriched red blood cells kofnasquirrelmcdcey(padI)andimmunct . . prwpltatknz of culture supematant from Homokgy to P. m --c ruptured schlzonts that had been metaboli- 235 kDa protein tally 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) con- 4-l-- --

diions. The RWIIRW-2 ‘&&let binds ph&yJ is co-p=ipbfed (panel 2).

- remamsasa monomer under NR conditiOme. As previously mported. the Mamzolte -.y&$

Inti-FvRRP-I antibodies as anatysed by western immunoblor. do not crossreact with -cz

FvRW2(Ref.&The MOkDabandinpanel I ‘.‘::..” .;. . . .:;y!: .- ..,_ ..,y . _-

reprerenoFvDElP.Thesmudgea~about i. \‘, ,..a; .-‘..\ “:;:I’;‘, .:

: l70-180!~Daisagelarcifactcausedbymigta- tion of a large amount of intact. nonreduced IgC and does not represent a specific imm~noprecipitated malaria protein. Pre-stained protein standards (GibcolSRL) and apo- ipoprotein 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 incer- thin disulphide bonds, consistent with experimental data indicating the present e of these covalent bondt; however, which of the I6 &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 multi- merit 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 auto- radiography (Fig. 2a). Reticulocyte-binding protein-l contains 16 cysteine residues, clustered into four re- gions, some of which could participate in the forma- tion of interchain, as well as intrachain disulphide bonds. Moreover, both of the PvRBP molecules tend to bind red blood cells in EBA analyses and co- precipitate 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 on the formation of interchain t ’ &&lphide bonds between monomers. Furthermore,

since PvRBP-2 often co-precipitates with PvRBP-1, it seems to associate with PvRBP-1 through noncova- lent 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. Some- 8Wtimes .@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 investi- gations, 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 sug- gest 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 be- tween 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 junc- i* 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~lesi merozoites 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 bind- ing, to satisfy the requirement of reticulocyte selec- tion. The predicted structure, position on the mero- zoite and adhesive feature of the PvRBPs together suggest a multifunctional role for these proteins. We have proposed that the PvRBPs target the reticulo- cytes for invasions. From this perspective, binding of these proteins would commit a merozoite to an appropriately selected host cell. When a vivax mero- zoite is released into the blood circulation, it is z&r- rounded by a preponderance of normocytes, cells it does not invade, but presumably comes in direct con- tact 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 li- gandfs). This necessary specific adhesion event could possibly serve the added functions of (11 stabilizing the weaker, reversible contact(s) between the mero- zoite and host cell that could be directly mediated by MSP-1 and/or other undassified adhesive surface pro- teins, 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 protein- bound) 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 for- mation 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 mero- zoites 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 irre- versible processes. Activation of the appropriate signaling pathway(s) by the requisite binding of the PvRBPs would ensure that the PvDBP is only re- leased in a timely manner. This scenario needs to be investigated furtherz’br. What form(s) or mecha- nism(s) of signal transduction might occur remains largely unknown. What is known, however, is that

Pcmsitofogy To&y, ~0’01. 12. no :, I996

Reviews --

staurosporine, an inhibitor of ser- ine/threonine kinases, inhibits entry of P. knowlesi merozoites into red blood cells, but not attachment or junction formationbs. This sug- gests that a phosphorylation path- way might affect a micro- 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 chemo- therapeutic interventions that could target crucial signaling pathways and inhibit parasite multiplication.

PvRBP analogdhomologs If, as postulated, the PvRBP

complex serves the multifunctional role of reticulocyte recognition and perhaps signaling for P. uivux merozoites, then from an evolu- tionary perspective it also is rea- sonable to propose that function- ally and structurally analogous merozoite molecules might be pres- ent in all species of Pfusmodiums. Plasmodium cytwmolgi, a reticulo- +-preferring closely related simian malaria, contains proteins homologous to PvRBP-1 and PvRBP-2. However, concurrent experiments showed that PvRBP gene probes did not cross-hybridize to the DNA of P. kndesi, P. fdcip- arum or P. berghei, even under conditions of lowered StringenCye. In addition, antibody crossreactivity was not observed when initial PvRBP antibody prepar- ations, which recognized a limited central portion of each protein, were tested for reactivity with these parasites. However, observations (see below) lend support to the hypothesis that a molecular complex comparable to the PvRBPs is present in other species of Plasrwditim.

Fig. 3. cartoon illunmi,q the pliit of the pkamocfii~vi~~~ merozoite. T&n to 16 P. vivm merozoites are produced from each schizont. When released iktto the circulating blood, they may encounter 50-200 normocytic eryth- rocytes before ‘bumping into’ a reticu- locyce. Logical reasoning would indi- cate that homing co a reticulocyte would require a specific mechanism, such as a reticukxyce receptor, ‘in place’ at the time merozoices are released from an infected cell. The effi- ciency of P. vimt sekdon and invasion Df reticuloqteN7 is attested by the fact thas during the acute phase of an mfectkm in either rum-immune humans or spknectomized monkeyx P. vivox multiplie+s by eight- to l2-fold between tach 48 h cycle of growth.

First, a precedent exists in earlier studies of P. knowlesi that would suggest that other proteius function apically prior to the interaction that leads to junction formationQ. In the absence of the erythrocyte Duffy glycoprotein, the merozoite can remain api- tally attached via long fiber-like macromolecules. What comprises these fibrils? These long-ranging fib- rils would be consistent with the large, hydrophilic and a-helical molecular structure, predicted to be characteristic of the PvRBP for RBP-related) macro- motecules. Moreover, it is relevant to note that as the junction between the merozoite and erythrocyte membranes ‘moves’ around the merozoite as it enters the red blood cell, the apical pole remains ‘attached/ anchored’ via an electrondense region25.a that has the appearance of these fibrillar components. Second, rabbit antisera recently produced against recombi- nantly expressed partial segments of selected regions of PvRBP-1 recognize P. fdci~rum and P. knowlesi Pms;cchgy Tbdq, vol. 12. no. I. ! ‘)96

The most convincing evidence for the existence of analogous pro- teins among distantly related species of Plasmodium comes from the comparative analysis of the structure of a large-molecular- mass (235 kDa1 merozoite protein of P. yoelii tPy2351, which localizes to the rhoptries and has a roie in the invasion of normocytes@‘. Within the deduced peptide sequence encoded by the Py235 gene, there is a region. (of 500 amino acids) that exhibits a sig- nificant degree of homology to the partial sequence reported for PvRBP-2 (29.6% identity, 51.6% similarityl@ (Fig. 2b). The signifi- cance of this homology is sup- ported further by our more-recent analysis comparing the C-terminal

regions of both proteins. Approximately 40% of the PvRBP-2 sequence (3.75 out of 9.5 kbl had been reported previouslyR. This complete gene has since been cloned and analysed (M.R. Galinski, unpub- lished). Interestingly, there is no additional significant sequence homology between this and thr Py235, but structurally they appear to be related. Both proteins contain aproximately 400 amino acids C-terminal to the previously noted homology, and both have a putative transmembrane domain that is immediately preceded by a set of short repeated amino acid motifs and followed by an apparent cytoplasmic domain (Fig. 2b). Thus, Py235 and PvRBP-2 appear to belong to the same protein family. As these two highly diverged species seem to manifest a gene product that is functionally and structurally similar, it is rea- sonable to assume that a related gene is present in P. fnlcipwum and in other species of malaria.

Alternative receptors fulfill multiple requirements It has become evident over the past decade that

P. fufciprunz is capable of utilizing alternative recep- tors to reqnize and gain entry into red blood cell+r4. Not all strains of P. falcipnrunz require sialic acid or glycophorins for merozoite invasion and it is now believed that at least two merozoite receptors exist that are functional alternatives for EBA-175. Some strains of P. fnlcipnntm will invade erythrocytes that lack the appropriate glycophorin A ligand, but retain glycophorin B, and othel3: invade erythrocytes lacking

merozoites by IFA in a pattern that is identical to that seen on mero- zoites 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-mol- ecular-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.

25

* bQth glycophorin A and B ligands; cells lacking gly- cophorin 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, J3 and 71, all of which are expressed? This ‘redundancy’ provides an explanation as to why either anti-Duffy glyco- protein 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 tamicy x. ‘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

: 1 have 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 SNhether or 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 de- pendent 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 reticulo- qtes in vitro by 80-90% of the control cellsi”. How-

j.. &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 the squirrel a I ‘monkey is susceptible to P. vivnxll. In addition, the

I$u$y glycoprotein mAb only partially blocks inva- sion 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 pro- tein(s) that does not bind to the Duify glycoprotein, but may perform the function of the DBP. Redun- dancy 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 mero- zoite 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 mero- zoites, 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 func- tion 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 ex- pressed 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 para- sites 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 in- vasion 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 func- tional polymorphism in the PvRBPs. Both of the PvRBP genes are present as single-copy genes. Al- though 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 ‘pref- erence’, which, at best (for P. vi>.!i:. or P. cynomolgi), can be defined as restriction of early ring-stage para- sites to morphologically identifiable reticulocyte9. We also have found that the restriction enzyme pat- tern 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 creat- ing alternative binding domains (or, perhaps, altema- tive 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 in t 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 anti- body dependent toxicityP5, or interference with biological processes such as merozoite protease ac- tivity%. 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 identifl- cation 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 po- tepiiai candida@ for develo ent of ti anti-merozbte

R” vaccine that mterfexes wit receptor-ligand interac- tlons. In the case of the PvRBP complex, the reticulo- q&-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 or the binding of PvDBP has P been determinedsz. However, an efficient method of production of this domain, which retains the struc- tural basis for both adhesion and lmmunogenicity, as well as the detailing of the finer structural determi- nants 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 investi- gated for P. vivax or P. falcipurum, and the putative rece

et?! tor function of the AMA-l protein familyw58

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 mero- zoite 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 read- ily 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 infec- tions due to the promotion of junction formation and Parasrcology Today, vol. 12. no. I, I996

invasion. An anti-PvRBP vaccine could also, poten- tially, 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 high- light 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 (struc- tural and functional) in the receptor molecules, and the relationship of this polymorphism to immuno- genicity. 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

49 binding and, thus, neutralization of

invasion, slmi r 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 agglu- tinating and cytophilic antibodies, may help to achieve a greater maximum and broader protection.

Concluding remarks With five P. vivax merozoite vaccine candidate hind-

ing proteins now under investigation, there clearly has been significant progress in the field of P. vizvnc vac- cine 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 cas- cade of molecular events that seems to occur prior to P. vivax merozoite invasion of reticulocytes tie. RBP binding preceeds DBP binding), however, we cer- tain] P. f~ Y

would include RBP-like analogs to the list of ciprutn candidates.

We still have a fair way to go towards understand- ing 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. Nutwith- standing, 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 pre- liminary 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 relation- ship to their host environment. As we uncover more detail about the malaria parasite and its biological processes, we will be in a considerably stronger pos- ition to devise more effective immunogenic compounds for vaccimtto!?.

Whitehead Award ;uld JWB 1s an I!ma T. Hlrscnl ‘Tr,;t crholx

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66 Schofwld, L. and Hack&, F. (1‘83) Signal transduction in host cells by a glycosylphosphatidylinositol toxin of malaria para. sites. 1. E.rp. Mnl. 177, 145-153

!,i Coppl, R.L. (1992) Malaria - revealing the ties that bind. fmvsitofo~y Todt~y 8, 393-394

MI Ward, GE. et nl. (1994) Staumsportne inhibits invasion of erythrocytes by malarial memzoites. Exp. Rvtlsitol. 79,4HUY7

69 Keen, J.K. rt ,I/. (1994) A gene coding for a high molecular mass rhoptry protein of Pfnsmodium yoclii. Mol. Riohwr. Ptrm~ihl. 65,171-177

70 Mitchell, G.H. et ul. (1986) Invasion of erythmcytes by P/as- medium falciparum malaria parasites. Evidence for receptor heterogeneity and two receptors. Blood 67, 1519-1521

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75 Mason, S.J. PI n/. (1977) The Duffy blood group determinants: Role in the susceptibility of human and animal erythrocytes to Plasmodium kaozult!si malarta. Br. J. //~?c~mltd. .36. 327-335

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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

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X3 Heldin, C-H. (1YYS) Dimeritation of cell surface receptors in signal transduction. Cc,// Ho, 213-223

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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

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In your February Pt~~~skology Today There will also be an insert of the Author and Subject Index for Parasitology Toddy 1995.

L 1

Erratum The correct ISBgV for the book Parasites in Human Tissues, reviewed in Parasitology Todq I I, p. 440.

1995. is 0 89189 379 2.

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