WO2001013923A1

WO2001013923A1 - Malaria gpi anchors as vaccines anti-parasitic drugs and for use in diagnostics

Info

Publication number: WO2001013923A1
Application number: PCT/US2000/022876
Authority: WO
Inventors: D. Channe Gowda; Eugene A. Davidson
Original assignee: Georgetown University
Priority date: 1999-08-20
Filing date: 2000-08-18
Publication date: 2001-03-01

Abstract

This invention relates to compositions and methods for treating or preventing malaria in host organisms, especially malaria in humans. The methods for treating or preventing malaria involve inhibiting or blocking the action or pathology mediated by a Plasmodium glycosylphosphatidylinositol (GPI). The invention also provides a kit for diagnosing whether a subject has been exposed to malaria.

Description

MALARIA GPI ANCHORS AS VACCINES

ANTI-PARASITIC DRUGS AND FOR

USE IN DIAGNOSTICS

FEDERAL FUNDING As research performed in this specification was supported, in part, by a grant from the NIAID (RO1 Al 41139), the government may have rights in the invention.

FTELD OF THE INVENTION

The invention relates to compositions, vaccines and methods for inhibiting, diagnosing and treating malaria and/or the physiological effects associated with infection in a subject by a malaria inducing parasite, e.g. Plasmodium, which expresses a glycosyiphosphatidylinositol (GPI). The new compositions and methods are directed toward inhibiting biosynthesis of a Plasmodium GPI, inhibiting its ability to induce malaria associated pathology, using GPI for diagnosis of malaria, and GPI- containing vaccines.

BACKGROUND OF THE INVENTION

Protozoan parasites cause devastating human suffering and death over time, and their global impact remains enormous in malaria holoendemic areas. Plasmodium alone causes several hundred million new cases of malaria annually (John R. David et al., "Molecular biology and immunology of parasitic infections," IN HARRISON'S PRINCIPLES OF INTERNAL MEDICINE 865-871 (13^th ed., Isselbacher et al., eds. 1992)). The problems with fighting malaria is compounded by development of drug resistance in these parasites, for example chloroquine resistance in Plasinodium falciparum.

I. Malarial Treatments, Vaccines and Diagnosis Malaria is a protozoan disease and is the most important of the human parasitic diseases, as it causes 1 to 3 million deaths per year. The causative agent of malaria is the protozoa, Plasmodium. Human malaria is caused by four species of Plasmodium: P. falciparum, P. malariae, P. vivax and P. ovale, with P. falciparum being the most common and virulent. Typically, the parasite first enters the bloodstream through the bite of an infected female Anopheles mosquito, which, while feeding, injects small haploid sporozoites into the host animal. The sporozoites enter hepatic cells and undergo asexual multiplication fission (schizogony) thereby producing merozoites. The merozoites are released from the liver cells whereupon they attach to and penetrate erythrocytes. Once inside an erythrocyte, the Plasmodium begins to enlarge as an uninucleate cell, termed a trophozoite. The trophozoite nucleus divides asexually to produce a schizont, which has 8 to 32 nuclei. The schizont divides and produces mononucleated merozoites. Eventually, the erytbrocyte lyses, releasing the merozoites into the bloodstream to infect other erythrocytes. This erythrocytic stage is cyclic, repeating itself every 48 to 72 hours or longer, depending on the species of Plasmodium. Occasionally, merozoites differentiate into macrogametocytes and microgametocytes, which do not rupture the erythrocyte. When these gametocytes are ingested by a mosquito, they develop into the female and male gametes, respectively, and will begin the process to form sporozoites anew. See LANSING M. PRESCOTT ET AL., MlCROBOLOGY 758-761 (1990).

Upon entry into the mosquito, the complicated sexual cycle begins in the 25 midgut of the mosquito. The red blood cells disintegrate in the midgut after 10 to 20 minutes. The microgametocyte continues to develop through exflagellation, and releases eight highly flagellated microgametes. Fertilization occurs with the fusion of the microgamete into a macrogamete. The fertilized parasite is known as a zygote, which develops into an ookinete. The ookinete penetrates the midgut wall of the mosquito and transforms into the oocyst within which many small sporozoites form. When the oocyst ruptures, the sporzoites migrate to the salivary gland of the mosquito via the hemolymph. Once in the saliva of the mosquito, the parasite is now ready to be injected into a host.

Anti-malaria parasitic drugs can be categorized by the stage of the parasite that they affect and the corresponding clinical objective (James W. Tracy et al, "Drugs used in the chemotherapy of protozoal infections — Malaria," IN GOODMAN & GILLMAN'S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS 965-985 (9^th ed. 1996). Most antimalarial drugs were developed on the basis of their action against asexual, erythrocytic forms of the malaria parasite, which are responsible for the clinical illness in a patient. Highly effective chemotherapeutic agents against erythrocytic forms of the malaria parasite in this category include chloroquine, quinine, quinidine, amodiaquine, mefloquine and halofantrine. Pyrimethamine, sulfonamides, sulfones and tetracyclines share this property but are slower acting. Other anti-malarial agents include tissue schizontocides (e.g., chloroguanide, chloroquine, and primaquine), blood schizontocides (e.g. antimalarial alkaloids or antimalarial endoperoxides), gametocytocides (e.g., chloroquine, quinine, and primaquine) and sporontocides (James W. Tracy et al., "Drugs used in the chemotherapy of protozoal infections — Malaria."). These antimalarial agents can be used either prophylactically to prevent a malaria infection, or as a method of controlling the conditions associated with a malarial infection in a host. A successful vaccine against Plasmodium has yet to be identified, although several anti-malarial vaccines have been prophetically described.

For example, U.S. Patent No. 4,957,738 (1990) to Patarroyo describes a mixture of synthetic peptide compounds to induce antibody production against late stages of malaria caused by P. falciparum. But the SPf66 malaria vaccine, comprising a synthetic polypeptide based on pre-erythrocytic and asexual blood-stage proteins of P. falciparum, has proven to have low efficacy in vaccinated subjects (Tanner et al., Schweiz. Med. Wochenschr. 126: 1210-5 (1996); Alonzo et al., Med. Trop. fMars 55(4 Supp): 41-6 (1995); and Acosta et al., Trop. Med. Tnt. Health 4: 368-76 (1999)). Another malarial vaccine used the protein, Pfs48/45, but it failed to block parasite transmission in a standard mosquito membrane feeding assay (Milek et al., Exp. Parasitol. 90: 165-174 (1998)). A malaria sporozoite vaccine candidate, RTS,S, formulated with an oil-in-water emulsion plus the immunostimulants monophosphoryl lipid A and the saponin derivative QS21 (vaccine 3), was shown to be somewhat protective against malaria (Stoute et al, J. Infect. Pis. 178: 1139-44 (1998); and Stoute et al, N. Erigl. J. Med. 336: 86-81 (1997)). The addition of a glycosyiphosphatidylinositol (GPI) anchor to a P. falciparum polypeptide, such as circumsporozoite protein (CSP), has been demonstrated to elicit a higher immune response than the corresponding polypeptide lacking a GPI anchor. A polypeptide with a GPI anchor attached has been proposed for use in a vaccine for inducing an immune response against the polypeptide (WO 96/34105 by Fasel et al.).

U.S. Patent No. 5,151,445 (1992) issued to Welphly et al, describes a method of inhibiting Trypanosomal parasites by inhibiting the biosynthesis of trypanosomal GPI anchors by incorporating an oxy-substituted fatty acid analog in place myristate, as myristate analogs are toxic to humans. However, the use of GPI alone to raise an immune response, agents for inhibiting malarial GPI biosynthesis, and methods of inhibiting GPI activity in a Plasmodium infected host have not been described.

U.S. Patent No. 5,853,739 (1998) to Kaslow et al, describes a transmission blocking vaccine. The patent relates to a method of creating antibodies to P. falciparum protein, Pfs25, which purportedly blocks transmission of the parasite from the host to the mosquito vector.

U.S. Patent No. 5,720,959 (1998) to Holder et al. describes an anti malarial vaccine comprising antigenic peptides from the merozoite surface protein 1 (MSP1) of P. falciparum. Another patent, U.S. Patent No. 5,393,523 (1995), issued to Knapp et al. describes a protective 41 kD antigen (e.g., SHARP or PfHRPII) of P. falciparum rich in alanines and histidines.

U.S. Patent No. 5,229,110 (1993) issued to DuBois et al., describes a malarial vaccine comprising at least one polypeptide extracted from a schizont form of Plasmodium, wherein the polypeptides range in weight from 70,000-120,000 daltons.

U.S. Patent No. 4,466,917 (1984), provides antisera and monoclonal 20 antibodies against the sporozoite stage of the parasite using a 44 kD plasmodium antigen. Notwithstanding the above-identified patents, Applicants are unaware of any malarial vaccine that has proven effective for wide scale use in endemic regions.

II. Glycosylphosphatidylinositol (GPJ)

GPJs are a class of glycolipids common to all eukaryotes, and were first described from Trypanosoma brucei. Recent evidence suggests that GPIs may mediate signal transduction within cells. For example, GPI-anchored proteins are physically associated with the src-family protein tyrosine kinases (PTKs) yH, lck, fgr, lyn and hck (Tachado et al, Proc. Nat Acad. Sci. USA 94: 4022-27 (1997)). Some have suggested that because the GPI biosynthetic machinery may be sufficiently different between mammalian and protozoa, it may represent a target for anti- protozoan chemotherapy (Sutterlin et al., EMBO J. 16: 6374-83 (1997); Heise et al., Braz. J. Med. Biol. Res. 27: 233-8 (1994); and Tiede et al., Biol. Chem. 380: 503-523 (1999)).

The nature and extent of glycosylation in Plasmodium βaciparum, one of the four agents causing malaria in humans, has long been controversial (Gowda et al., Parasitol. Today 15: 147-152 (1999)). Recent studies have indicated that P. falciparum has a very low N-glycosylation capability and O-glycosylation is either absent or present at extremely low levels, whereas glycosylphosphatidylinositol (GPI) anchor modification is common and is the major carbohydrate modification of the parasite proteins (Gowda et al., 1999). Unlike O- and N-glycosylation, only one GPI anchor is found per protein molecule, attached to the C-terminal amino acid of the protein through an ethanolamine phosphate (Gowda et al., 1999).

Parasitic GPIs from P. falciparum are sufficient to initiate signal transduction when added alone to host cells, as agonists (Tachado et al., 1997). Moreover, parasitic GPIs possess a dual capacity as agonist and as second messenger substrates (Tachado et al., 1997). Plasmodium GPIs induce the expression in host cells of inducible nitric oxide synthase (iNOS), tumor necrosis factor a (TNFα), interleukin-1 (IL- 1 ) and adhesins, which activate various signaling pathways in host cells. This in turn may cause erythrocyte sequestration, hypoglycemias, triglyceride lipogenesis and immune dysregulation. Some have reported that GPI is the Plasmodium toxin, which can cause the lethal complication of malaria known as cerebral malaria.

Administration of anti-GPI antibodies or immunization against the GPI or various nontoxic analogues have been proposed as clinical protection against malarial disease [Schofield et al, J. Exp. Med. 177: 145-53 (1993)]. Monoclonal antibodies to GPI neutralize NO induction by malaria parasites [Tachado et al., J. Immunol. 156: 1897-1907 (1996)]. Several monoclonal antibodies to GPI were prepared which inhibit induction of TNFα by total schizont extracts, further indicating that GPJ or a serologically related structure is the dominant TNFcc-inducing agent produced by P. falciparum parasites [Schofield et al, Annals Trop. Med. & Parasitology 87:-617-26 (1993)].

In P. falciparum, several functionally important proteins of the intraerythrocytic stage are modified with GPI anchor moieties. These include MSP-1, MSP-2, MSP-4, p71, the 55 kD merozoite rhoptry antigen, 102 kDa transferrin receptor and a 76 kD seine protease (Gowda et al, 1999).

Additionally, malarial GPI is yet to be fully characterized. The glycan 20 cores of the GPI anchor moieties contain a conserved trimannosylglucosaminyl moiety with an additional α 1,2-linked mannose attached to the mannosyl residue distal to the glucosamine residue. The GPI anchor moieties of these proteins are described as containing myristic acid and palmitic acid on their inositol or a diacylglycerol moiety depending on the protein (Bamwell et al, Exp. Parasitol. 91: 238-49 (1999)). Gowda et al were the first to report the presence of acid-labile, unusual substituents on the terminal 1,2-linked a-mannosyl residues (J. Biol. Chem. 272: 6428-6439 (1997)). However, the precise glycosyl structure or structures of Plasmodium has yet to be fully elucidated. The scientific communities believe that these parasites contain a variety of GPI precursors and intermediates not in association with proteins, and that the glycosylphosphatidylinositols of this type differ in polarity and hydrophilicity through the addition of ethanoalamine and sugar groups. These other potential GPIs may also contribute to the pathophysiological response of the host when released during merogony or cell death (Schofield et al, 1993).

GPI anchors of P. falciparum proteins have Ma -GlcN cores with substituents that are susceptible to the conditions of nitrous acid deamination on the terminal mannose (Man) residues (Gowda et al, 1997). Specifically, the glycan cores of the parasite protein GPJ anchors have the following structure:

O

II Protein-NH-CH₂-CH₂-O-P-O

I o

\

6 Manαl→2Man l→4GlcNαl→R₂

2 / _Y-Manccl

where X represents an unidentified substituent. R₂ represents the 25 phosphatidylinositol (PI) and a fatty acid component of the GPI molecule which is likely cis-vaccenic acid.

Recently, the inventors of this application reported that the addition of mannosamine (ManN), a known inhibitor of GPI anchor biosynthesis in eukaryotic cells, to a culture comprising synchronous cultures of ring stage intraerythrocytic P. falciparum, nearly completely stopped parasite development at the early trophozoite stage, and these parasites died in the first culture cycle (Naik et al, "#78- Glycosylphosphatidylinositol (GPI) Anchor Biosynthetic Pathway is Essential for the Growth and Development of Intraerythrocytic Plasmodium falciparum, (Abstract)" Glycobiology 199: 78 (1998)). Mannosamine has been demonstrated to effectively inhibit the biosynthesis of 5 GPI in Leishmania mexicana (Field et al, J. Biol. Chem. 268: 9570-7 (1993)). A terpenoid lactone, YW3548, was found effective at blocking the addition of the third mannose to the intermediate Man2-GlcN-acyl-PI, which was proposed as a target for anti -protozoan chemotherapy (Sutterlin et al, EMBO J. 16: 6374-83 (1997)). However, to the best of the inventors' knowledge, use of a compound that inhibits Plasmodium GPJ biosynthesis in vivo to treat or prevent malarial infection in a subject has never been reported. Nor are Applicants aware of the successful use of GPI or immunogenic fragments thereof in a vaccine preparation to effectively prevent malaria in a subject.

OBJECTS AND SUMMARY OF THE INVENTION

Despite massive investment aimed at developing a malaria vaccine and methods of effectively treating malaria, and the advances in understanding the biology and immunology of malaria, a safe, effective and long-lasting vaccine or curative treatment has not been discovered to the knowledge of the inventors.

It is an object of the invention to provide a method for treating or preventing malaria pathogenesis in a subject comprising the step of administering a therapeutically effective amount of an agent which inhibits Plasmodium glycosylphosphatidylinositol (GPI) biosysnthesis or which inhibits the immune response induced by Plasmodium GPI. GPI is apparently important to parasite survival. Plasmodium GPIs contemplated for use in this method include those of P. falciparuin, P. malariae, P. vivax and P. ovale. These GPI structures can be derived from a variety of Plasmodium proteins modified with a GPI, including MSP-1, MSP- 2, p71, the 55 kDa merozoite rhoptry antigen, 102 kDa transferrin receptor and a 76 kDa serine protease.

The malarial GPIs proposed for use in vaccines which comprise another object of the invention include that of:

wherein n can be 12, 14, 16, 18 or 20 and is preferably 16; and Ri is Cι₅H₃₁ (major) or Cι₃H₂₇ (minor); and R₂ is C₁₇H₃₃ (major) or C H₃₁ (minor).

Also contemplated is the use of GPI fragments in vaccines. These GPI fragments include:

Ino-O-C=O

(i) o=p-σ R,

I

O

I CH_rCH-CH₂

I I

0 0

1 I C=O CO

R* R ^•3 or

GlcNαl- Ino-O-C=0

I I (π) o=p-o- R,

I o

I

CH₂-CH-CH₂

I I O O

I I

C=0 C=O

I I ** ^RJ wherein Rj, is C₁₅ H_3! or C₁₃H₂₇, R₃ is C₎₇H₃₃ or Cι₇H₃₁ and R₂ is CH₃(CH₂)_n, wherein n is 12, 14,16, 18 or 20, and wherein n is preferably 16. R_\ may also be myristic acid or palmitic acid. R₃ may also be oleic, cis-vacceric or linoleic acid. Additional glycosyl residues can be added to the immunogenic fragment, e.g., at the end of GlαNαl. These glycosyl residues may be substituted or unsubstituted and include by way of example mannose, glucose, or galactose. Potential substitution groups include, by way of example, protecting groups such as phosphate, sulfate or acetyl. The number of glycosyl groups, substituted or unsubstituted may vary from about 1 to 50, more preferably about 1 to 5, wherein the terminal glycosyl, e.g., mannose is preferably substituted.

An example of such structure is set forth below:

Man 1 → 4-GlcN-Ino-O-C = O

I I

O

CH₂-CH-CH₂

O O

CO CO

R₂ R₃

In the above structure, Rj, R₂ and R₃ are as previously defined.

It is another object of this invention to provide pharmaceutical compositions which inhibit the biosynthesis of GPI. Such compositions may include agents, such as mannosamine, GlcN, benzyl-α-2-amino-2-deoxy-glucosamide, phenyl-α-2amino- 2deoxy-glucosamide and aryl-2-amino-2-deoxy-glycosamide or other compounds which effectively inhibits GPI-biosynthesis.

Another object of the invention is to provide pharmaceutical compositions which contain compounds that produce a protective immune response in a host or compounds which interfere with GPI biosynthesis thereby killing the parasite. Another object of the invention is to diagnose subjects, preferably humans, to determine whether they possess the ability to mount a protective immune response against a malaria infection.

BRTEF DESCRIPTION OF THE DRAWINGS Figure 1. SDS-PAGE fluorograph of [³H]-glucosamine labeled proteins of P. falciparum. Parasites were metabolically labeled with [³H]-glucosamine, and the cell lysates electrophoresed under non-reducing conditions on 5-20% SDS- polyacrylamide gradient gels, and then fluorographed. Panel A: FCR-3 strains labeled at different stages of the intraerythrocytic development. Lane 1 , labeled for 16 h, immediately after synchronization with sorbitol (rings); Lane 2, labeled for 6 h, 18 h after synchronization (rings and trophozoites); Lanes 3 and 4, labeled for 6 h, 24 h after synchronization (mainly trophozoites and schizonts); Lane 5, labeled for 6 h, 30 h after synchronization (trophozoites and schizonts); Lanes 6 and 7, labeled for 6 h, 36 h after synchronization (trophozoites and mainly schizonts). Panel B: D6 strain (Lane 1), W2 strain (Lane 2) and NF54 strain (Lane 3) labeled for 16 h after synchronization (rings).

Figure 2. Analysis of Sera for Presence of Anti-GPI Antibodies. Infants less than one year of age (upper panel) have few antibodies. Siblings of these infants ranging in age of 7-8 years are more likely to have anti-GPI antibodies in their sera.

Adults living in endemic areas have the highest likelihood of having anti-GPI antibodies in their sera.

Figure 3. The structures of P. falciparum GPIs as determined by mass spectrometry. Fig. 3A shows a HPTLC fluorogram of the total free GPIs (lane 1), purified, matured free GPIs (lane 2), total free GPIs (lane 3), and total protein-linked

GPIs (lane 4). Each lane contains 200 ng of GPIs plus 20,000 of corresponding

[³H]glucosamine -labeled GPIs. Fig. 3B shows a representative mass spectrum of the purified free GPIs that contain (M-H) ions at m/z 1977.3, 2005.3, 2061.3 and 2089.3. Fig. 3C shows a representative mass spectrum of phospholipase A₂-treated free GPIs showing two sets of (M-H)^" ions; one set representing intact GPIs and the other is due to GPIs in which the acyl substituent at the C2 of glycerol has been removed. Fig. 3D shows a representative mass spectrum of the protein-linked GPI Fraction I showing the (M-H)^" ions at m z 2092.3, 2120.4 and 2148.2. The mass spectrum of Fraction π contained (M-H)^" ions at m/z 2147.3 at significantly higher proportions compared to that of Fraction I, in addition to an ion at m z 2175.3. Fig. 3E shows the cleavage sites for the attachment of GPIs in P. falciparum merozoite surface proteins. Fig. 3F shows a representative mass spectrum of phospholipase A₂-treated protein-linked GPIs showing (M-H)^" ions at m/z 1827.8, 1855.8, 1883.8 and 1910.8 that are due to GPIs in which the acyl substituent at the C2 of glycerol has been removed.

Figure 4. HPLC and HPTLC purification of P. falciparum GPIs. A, The parasite GPIs (10 μg plus 400,000 cpm of [³H]GlcN-labeled GPIs) were chromatographed on a 4.6 X 250 mm C₄ reversed phase HPLC column with a linear gradient of 20 to 60% aqueous 1-propanol. Upper panel, analysis of parasite GPIs: Fractions (1.0 ml) were collected and ³H activity in 5 μl aliquots measured (•); 0.5 μl aliquots assayed by ELISA for immunoreactivity with Kenyan adult sera (O). Lower panel, analysis of glycolipids from control erythrocyte membrane debris obtained from 4 ml packed erythrocytes (•), and those of delipidated, pronase-digested erythrocyte ghosts from 2 ml packed erythrocytes (O). One-μl aliquots were assayed for immunoreactivity with Kenyan sera. Extracts of total erythrocyte lysate were similarly analyzed (not shown). B, Fluorograms of the [³H]GlcN-labeled GPIs chromatographed on Silica Gel 60 HPTLC plates using CMW (10:10:2.5, v/v/v). Lane 1, total free GPIs before HPLC; lane 2, HPLC-purified matured free GPIs; lane 3, total free GPIs (different preparation from those in lane 1; obtained by culturing parasites in regular medium after replacing medium with radiolabeled precursor to maximally convert intermediates into matured GPIs); lane 4, HPLC-purified, protein- linked GPIs. Each lane contains 200 ng of GPIs plus 20,000 cpm of [³H]GlcN- labeled GPIs.

Figure 5. Mass spectrometry analysis of P. falciparum GPIs. HPLC- and HPTLC-purified GPIs were analyzed by matrix-assisted laser-desorption/ionization time of flight mass spectrometry in negative or positive ion mode. A, Negative ion mass spectrum of the purified free GPIs. B, Negative ion mass spectrum of phospholipase A₂-treated free GPIs showing (M-H)^" ions of unconverted GPIs and

GPIs lacking substituent at sn-2 position. C, Positive ion mass spectrum of inositol- acylated glycan moiety released by HF-treatment of GPIs. D, Negative ion mass spectrum of inositol-acylated glycan moiety released by HF-treatment of GPIs. E,

Negative ion mode mass spectrum of the protein-linked GPI Fraction I (Fig. IB). It can be seen from these results that the mass spectrum of Fraction II (Fig. 4B) contained (M-H)^" ions at m/z 2147.3 at significantly higher proportions compared to that of Fraction I, in addition to an ion at m/z 2175.3 (not shown). F, Negative ion mass spectrum of phospholipase A₂-treated protein-linked GPIs showing (M-H)^" ions of unconverted GPIs and GPIs lacking substituent at sn-2 position. G, The cleavage sites for the attachment of GPIs in P. falciparum merozoite surface proteins.

Figure 6. P. falciparum GPIs contain C 18:1 fatty acid at sn-2. The parasites were metabolically labeled with various [³H]fatty acids. Free GPIs were isolated, treated with bee venom phospholipase A₂, and analyzed by HPTLC. -, untreated GPIs; +, phospholipase A₂-treated GPIs.

Figure 7. The proposed structures of P. falciparum GPIs. The sn-\ position contains saturated fatty acyl substituent (C14:0 to C22:0), the sn-2 position has predominantly C18:l, and C-2 of inositol contains palmitic (major) and myristic (minor) acids. Figure 8. ELISA for naturally elicited anti-GPI antibodies in human sera. Purified GPIs were coated on to 96-well microtiter plates either at indicated amounts per well (A) or at 2 ng per well (B). The wells were blocked with 0.5% casein in TBS, pH 7.4 (TBS-casein), and then incubated with human sera, 1 :100 or serially diluted with TBS-casein, 0.05% Tween-20. After washing the plates, the bound antibodies were detected with HRP-conjugated goat anti-human IgG (H+L chains) using ABTS substrate. •, Kenyan adult sera 1; , Kenyan adult sera 2; , Kenyan adult sera 3; O, USA adult control serum.

Figure 9. Inhibition of anti-GPI antibody binding to GPIs by phospholipids.

The purified GPIs were coated on to microtiter plates (2 ng), blocked with TBS- casein, and overlaid with representative Kenyan sera (1 :200 diluted) incubated with the indicated phospholipids and purified GPIs. The bound antibodies were measured by HRP-conjugated goat anti-human IgG (H+L chains) using ABTS substrate. Shown is the date from a representative of 10 different sera analyzed. , without inhibitor (control); D, 2.5 ng; D, 5 ng; D, 10 ng; Q 20 ng.

Figure 10. HPTLC immunochromatogram of P. falciparum GPIs. GPIs (100 ng each) were chromatographed on HPTLC plates, blocked with 1% BSA, and incubated for 2 h in 1 : 100-diluted sera. The bound antibodies were detected with ¹²⁵I- labeled goat anti-human IgG (5 μCi/ml). Lanes 1 and 2, reactivity of free and protein-linked GPIs (before HPLC fractionation) with Kenyan adult sera; lanes 3 and 4, free GPIs and protein-linked GPIs, respectively, treated with control USA adult sera; lanes 5 and 6, Pis from bovine liver and soybean, respectively; lane 7, PG; lane 8, CL, treated with Kenyan adult sera. Shown are representatives of 10 Kenyan and USA adult sera analyzed. Parasite lipids other than GPIs and their intermediates, extracted with chloroform methanol (2:1, v/v), and glycolipids from control erythrocytes were completely nonreactive to Kenyan sera (not shown). Figure 11. Age-dependent anti-GPI antibody response in people living in malaria endemic area. Sera from a cohort of children and adults were analyzed by ELISA (Fig. 10). The mothers and children were followed every two weeks, and clinical parameters, temperature and illness recorded. Blood samples were taken monthly, and parasite and hemoglobin density were measured. Experiments were performed in duplicates. An antibody level (OD) greater than the mean of USA adult control sera plus two standard deviations was considered positive. A, Percent individuals in the following anti-GPI antibody responder categories: negative at both 1-3 month-spaced time points sampled = negative ( ) , positive at one time point = intermittent ( bar), and positive at both time points = positive ( ) . n>40 in each age group between -0.5 and 3.5 years, and n - 100 and 50 in the 7-8 years and 20-25 years age groups, respectively. Ax² test found that the antibody responder category was different among age groups (pθ.001). B, Mean hemoglobin (g dl, ), temperature (-29.5°C), and anti-GPI antibody level [(log₁₀ (OD+1)] (•) for the indicated age groups. ANOVA found that the anti-GPI antibody level, hemoglobin and temperature were different among age groups (p<0.0001).

Figure 12. Serum anti-GPI antibody response and resistance to malaria in children 0.5-3.5 years of age. A General Linear Model (GLM) was used to investigate the correlation between anti-GPI responder category and temperature. (A) or hemoglobin level (B), while controlling for age and parasite density. A, Mean hemoglobin (g/dl) for 0-3.5 years-old negative ( ) , intermittant ( ), and positive

( ) anti-GPI antibody responder categories. B, Mean temperature (°C) for 0-3.5 years-old negative ( ), intermittant ( ), and positive ( ) anti-GPI antibody responder categories. The anti-GPI antibody responder category and parasite density were independently associated with hemoglobin (p<0.0147 and p = 0.0000, respectively). The associations were independent of the non-significant association between age and hemoglobin (p = 0.0581). Antibody responder category and parasite density were independently associated with temperature (p = 0.0012 and p = 0.0001, respectively). Age was not found significantly correlated with temperature (p>0.0581).

Figure 13. Fatty acid composition of P. falciparum GPIs produced according to the invention. The HPTLC-purified total GPIs (Fig. 4B lane 2), inositol-acylated glycan moiety obtained by HF treatment of purified GPIs, and 1-O-monoacyl-glycerol moiety obtained by HF treatment of phospholipase A₂-treated GPIs (lyso-GPIs) were saponified with methanolic KOH at 65°C for 1 h. The solution is dried under nitrogen, acidified to pH 4 by 1 M HC1, fatty acids were extracted with 1 -hexane and converted to their methyl ester with diazomethane. The resulting methyl esters were analyzed by GLC-MS. Fatty acids released by treatment of the purified GPIs with phospholipase A₂ were also similarly analyzed. (The numbers in the parenthesis represent approximate mole proportions, not corrected for response factors.)

DETAILED DESCRIPTION OF THE INVENTION It has been determined that GPI anchors are the only carbohydrate modification of P. falciparum proteins and also that glycan cores of GPI contain substituents that constitute unusual structural features. For example, it has been determined that Plasmodium GPI contains an unusual fatty acid, which appears to be antigenic. Moreover, mannosamine (ManN), an inhibitor of GPI biosynthesis, is lethal to P. falciparum. Accordingly, the subject invention includes at least the following: (1) vaccines containing Plasmodium GPIs or an immunogenic fragment thereof; (2) methods and compositions which inhibit GPI biosynthesis; (3) methods and compositions which block GPI-induced activity; and (4) GPI or an immunogenic fragment thereof for use as a diagnostic agent for determining whether an individual has been exposed to malaria; and (5) assays for detecting whether an individual is protected against malarial infection based on the presence or absence of antisera to GPI. I. Definitions

By "GPI" or "glycosylphosphatidylinositol" is meant that mature moiety located on all Plasmodium proteins, as well as the mature forms of GPI produced in excess by the parasite, which are not linked to a Plasmodium protein. By "substantially pure GPI" is intended a GPI or immunogenic fragment thereof that is substantially free of associated malarial proteins.

By "humanized antibody" is meant an antibody derived from a nonhuman antibody, typically a murine antibody, that retains or substantially retains the antigen- binding properties of the parent antibody, but which is less immunogenic in humans. This may be achieved by various methods, including (A) grafting the entire non- human variable domains onto human constant regions to generate chimeric antibodies; (B) grafting only the non-human complementarity determining regions (CDRs) into human framework and constant regions with or without retention of critical framework residues; and (C) transplanting the entire non-human variable domains, but "cloaking" them with a human-like section by replacement of surface residues. Such methods are disclosed in Morrison et al., Proc. Nail. Acad. Sci. USA 81: 685 1-5 (1984); Morrison et al, Adv. Immunol. 44: 65-92 (1989); Verhoeyen et al, Science 239: 1534-1536 (1988); Padlan, Molec. Immun. 28: 489-498 (1991); and Padlan, Molec. Immun. 31: 169-217 (1994), all of which are incorporated by reference in their entirety.

By "chimeric antibody" is meant an antibody containing sequences derived from two different antibodies, which typically are of different species. Most typically, chimeric antibodies comprise human and murine antibody fragments, and generally contain human constant and murine variable regions. By "bispecific antibody" is meant an antibody molecule with one antigen- binding site specific for one antigen, and the other antigen-binding site specific for another antigen. By "immunogenicity" or "immunogenic" is meant the ability of a targeting protein or other moiety, such as GPI to elicit an immune response (e.g., humoral or cellular) when administered to a subject.

It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. "Dosage unit form," as used herein, refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound is calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on: (A) the unique characteristics of the active compound and the particular therapeutic effect to be achieved; and (B) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.

By "therapeutically effective amount" or "prophylactically effect amount" or "dose effective amount" is meant an amount of an agent which modulates a GPI- mediated activity or otherwise inhibits or prevents replication and proliferation of Plasmodium in a subject.

By "GPI-mediated activity" or "GPI-induced activity" is meant any activity, condition, modulated by a biologically active form of GPI or a fragment thereof. For example, induction of iNOS or TNFα.

"Protective immunity" is the condition induced by the administration of a vaccine to a subject, wherein the susceptibility of the subject to infection by a particular pathogen is reduced.

By "susceptible subject" are those subjects, preferably animals, and more preferably mammals, in which Plasmodium is a pathogen. "Susceptible subjects" include living organisms in which an immune response can be elicited, e.g., mammals, and most preferably human. For example, susceptible human subjects include those visiting or living in areas that are prone to malarial infection, such as tropical locations. Subjects can further include primates, ungulates (e.g., bovine, ovine, caprine, porcine and the like), rodents (e.g., mice, rats, hamsters and rabbits), avians and other mammals infected by species of Plasmodium. Preferred examples of mammalian subjects include humans, dogs, cats, horses, cows, pigs, goats, sheep, mice, rats, and transgenic species thereof. By "susceptibility to infection" is meant the condition of being a host for a particular pathogen and of suffering injury from the disease caused by that pathogen.

The condition of "susceptibility to infection" encompasses a range of susceptibilities.

The degree of susceptibility of a particular subject to infection by a particular pathogen may be determined by calculating the LD₅₀ value for this pathogen. Subjects less susceptible to infection by a particular pathogen will have a higher LD ₀ for that pathogen than a more susceptible subject.

By "adjuvant" is meant any material that enhances the action of a drug or antigen.

By "pharmaceutical excipient" refers to any inert substance that is combined with an active drug, agent, or antigen for preparing an agreeable or convenient dosage form.

II. Vaccines and Methods of Prophylactically Treating Malaria

The vaccines of the present invention are contemplated to contain GPI or an immunogenic fragment thereof; which when injected in a host will confer protective immunity to the host against malaria. These vaccines may comprise necessary adjuvants, excipients and/or pharmaceutically acceptable carriers.

The vaccines contemplated by the inventors use a Plasmodium-deήved GPI as described or a fragment thereof to confer immunity in a host. The GPI fragment must be immunogenic and can include for example: phosphatidylinositol (PI), PI attached to the lipid portion of GPI, Pl-glucosamine, and Pis which further comprises fatty acid variants of various sizes. The GPIs contemplated for use in a vaccine include:

Also contemplated for use in the vaccines are immunogenic fragments of GPI which can include:

Ino-O-C=O

- I I

O=P-O- _t f O

1 I c=o c=o

I I

R- R₃

or GlcNαl- Ino-0-G=0

^— r 1

⁼μ— I— -

wherein Ri is Cι₅H₃₁ or C₎₃H₂₇, R₃ is Cι₇H₃₃ or Cι₇H₃₁, and R₂ is CH₃(CH₂)_n, wherein n is 12, 14, 16, 18or 20, but wherein n is preferably 16. Ri may also be myristic acid or palmitic acid. R₃ may also be oleic or linoleic acid. The GPI or GPI fragment containing vaccines can further comprise pharmaceutically acceptable carrier and excipients.

Carriers. One possible carrier is a physiological salt solution. Another pharmaceutically acceptable carrier is, for instance, the tissue culture fluid used for sustaining the cell growth, in which the parasites are released from the infected cells. Adjuvants. An adjuvant, and if desired one or more emulsifiers, such as

Tween and Span, may also be incorporated in the vaccine according to the invention.

Suitable adjuvants include, for example, aluminum hydroxide, vitamin E acetate solubilisate, sapoinin (e.g., ISCOMs), quil A, and more purified forms thereof; murayl dipeptide, mineral and vegetable oils (e.g., Bayol or Marcol52), DEAE dextran, non- ionic block copolymers or hposomes such as Novasomes, in the presence of one or more pharmaceutically acceptable carriers or diluents.

The vaccine according to the invention may be produced in a freeze-dried form. Suitable stabilizers are, for example, carbohydrates (e.g., such as sorbitol, mannitol, trehalose, starch, sucrose, dextran or glucose), proteins (e.g., albumin or casein), or degradation products thereof; and buffers (e.g., alkali metal phosphates). If desired, one or more compounds with adjuvant activity as described above can also be added. The vaccines according to the invention may be administered by intramuscular

(i.m.) or subcutaneous (s.c.) injection or via intranasal, intratracheal, oral, cutaneous, percutaneous/intradermal or intracutaneous administration. A very convenient way of administration is intradermal or intramuscular administration. Therefore, in a preferred embodiment, the vaccine according to the invention comprises a carrier that is suitable for intradermal or intramuscular application. A physiological salt solution is, for example a simple and suitable carrier for intradermal or intramuscular application.

In some instances, other materials, such as the adjuvants, emulsifiers and stabilizers mentioned herein, can be added to improve the performance and the stability of the vaccine.

The active compound, GPI or an immunogenic fragment thereof; by itself or in combination with other active agents, also may be administered by injection, oral administration, inhalation, transdermal application or rectal administration. Depending on the route of administration, the active compound may be coated with a material to protect the active compound from the action of enzymes, acids and other natural conditions which may inactivate the compound. A preferred route of administration is by intravenous (i.v.) injection.

To administer the vaccines comprising GPI or immunogenic fragments thereof described herein, it may be necessary to coat the vaccine agent or other agent with, or co-administer it with, a material to prevent its inactivation. For example, a Plasmodium GPI or GPI fragment can be administered to an individual in an appropriate carrier or diluent, co-administered with enzyme inhibitors or in an appropriate carrier or vector, such as a liposome. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Enzyme inhibitors include pancreatic trypsin inhibitor, diisopropylfluorophosphate (DEP) and trasylol. Liposomes include water-in-oil-in-water emulsions, as well as conventional Hposomes (Strejan et al., . Neuroimmunol. 7: 27-41 (1984)). Additional pharmaceutically acceptable carriers and excipients are known in the art. The active components may also be administered parenterally or intraperitoneally. Dispersions of the active compound can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain one or more preservatives to prevent the growth of microorganisms. Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the composition must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols, such as manitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating an active compound (e.g., a Plasmodium GPI) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying, which yields a powder of an active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

When the active compound is suitably protected, as described above, the 20 active agent may be orally administered, for example, with an inert diluent or an assimilable edible carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the therapeutic compositions is contemplated. All compositions discussed above for use with a Plasmodium-deήved GPI or immunogenic fragment thereof may also comprise supplementary active compounds in the composition.

The vaccines described above can be administered to the host species needing protective immunity by any of the conventional routes described above, and optionally at intervals. For example, two injections can be administered at a seven to thirty-five day interval. A suitable dose of the active agent may be GPI or an immunogenic fragment thereof in the range of 5-100 g/kg, or more preferably 5-10 g/kg. III. Compositions and Methods of Treating Malaria

The vaccines described above are preferred for prophylactically preventing infection by Plasmodium in a host. However, the instant invention also seeks methods of treating subjects infected with Plasmodium. The therapies contemplated by the instant invention include methods of (1) inhibiting GPI biosynthesis in a subject infected with Plasmodium, and or (2) inhibiting a GPI-mediated activity.

A. Inhibiting GPI Biosynthesis

Agents which inhibit GPI biosynthesis include mannosamine (ManN), GlcN, benzyl-α-2-amino-2-deoxy-glucosaminide, phenyl-α-2-amino-2-deoxy-glucosaminide and aryl-2-amino-2-deoxy-glycosaminide. These reagents can be administered alone or in combination with current anti-malarial therapies.

GPI synthesis inhibitors can be administered in pharmaceutical compositions and by any of the routes described above. Effective dosages (i.e., therapeutically effective amounts) of the agents which mediate GPI biosynthesis range from about 0.00 1 to about 30 mg/kg body weight, more preferably from about 0.01 to about 25 mg/kg body weight, and most preferably from about 0.4 to about 20.0 mg/kg body weight. Other dosages may be more efficacious and must be determined on a patient by patient basis. Factors influencing dosage include, but are not limited to, the severity of the disease; previous treatment approaches; overall health of the patient; age of the patient, etc. The skilled artisan is readily credited with assessing a particular patient and determining a suitable dosage that falls within the ranges, or if necessary, outside of the ranges, as needed.

B. Agents Which Modulate GPI-Mediated Activity

Another method by which Plasmodium infection in a subject can be treated to inhibit the agent which causes the pathology associated with malaria. As discussed above, Plasmodium GPI induces TNFα, as well as other cascades which produce malaria associated pathology. The instant invention also contemplates using agents which block GPI-induced pathology in a subject suffering from malaria. This can be employed by treating a patient using the vaccine described above to raise antibodies which bind to GPI. Alternatively, the inventors further contemplate the use of agents which block GPI-mediated activity or compete with GPI.

Agents contemplated which block GPI-mediated activity include antibodies or antibody fragments (e.g., Fab, scFv, F(ab')₂) which bind to a Plasmodium GPI. These antibodies can be raised via GPI vaccines, described above, or can be prepared recombinantly. The antibodies useful in treating malaria would be those which bind to GPI and thereby inhibit GPI-mediated activity.

Alternatively, agents which block or compete with a Plasmodium GPI can be used to treat malaria. Such agents can be administered in pharmaceutical compositions, such as those discussed above.

IV. Combination Therapy

It is another embodiment of the instant invention to use the methods and compositions described for treating malaria in combination with other known treatments for malaria. Several drugs are available for oral treatment, and the choice of drug depends on the likely sensitivity of the infection parasites. Despite recent reports regarding chloroquine-resistant P. vivax, chloroquine is still the recommended choice. Recommended regimens are detailed in Nicholas J. White et al., "Malaria and Babesiosis," IN HARRISON'S PRINCIPLES OF INTERNAL MEDICINE 887-896 (13^th ed 1994), which is herein incorporated by reference in its entirety.

The compositions for prophylactically treating malaria can also be considered in combination with a chemoprophylactic agent. Specific agents depend on the sensitivity of the local parasite. However, for P. falciparum, which is, present through most of the malarious world, mefloquine (250 mg weekly for adults) is the drug of choice. Doxycycline, dihydrofolate inhibitors (e.g., pyrimethamine and prognanil) and chloroquine (5 mg of base per kilogram per week, 300 mg maximum) can administered (Nicholas J. White et al, 1994). All of these agents are contemplated for co-administration with the compounds of the instant invention. V. Methods of Diagnosing Malaria

Another aspect of this invention is to use GPI, or an immunogenic portion thereof; to diagnose whether a subject has been exposed to malaria. The detection of parasite antigens present in a biological fluid (e.g., plasma) of a subject, such as GPJ or an immunogenic fragment thereof, can constitute a method of diagnosing all forms of malaria, regardless of the subject. Moreover, such diagnosis may alert medical personnel as to whether the person may be vulnerable to acute, chronic acute or chronic malaria infections.

Blood can be collected from a subject and tested for the presence of 10 antibodies which bind to a Plasmodium GPI or immunogenic fragment thereof. Such tests are exemplified in the Examples provided below.

Alternatively, one can mix labeled GPI or a labeled immunogenic fragment thereof with sera from a patient to determine the presence of anti-GPI antibodies. The methods of labeling GPI and immunogenic fragments thereof contemplated, including without limitation enzymatic conjugates, direct labeling with dye, radioisotopes, fluorescence, or particulate labels, such as liposome, latex, polystyrene, and colloid metals or nonmetals.

Examples of the types of labels which can be used in the present invention include, but are not limited to, enzymes, radioisotopes, fluorescent compounds, chemiluminescent compounds, bioluminescent compounds, particulates, and metal chelates. Those of ordinary skill in the art will know of other suitable labels for binding to GPI or an immunogenic fragment thereof. Furthermore, the binding of these labels to GPI or an immunogenic fragment thereof can be accomplished using standard techniques commonly known to those of ordinary skill in the art. One of the ways in which an assay reagent (generally, a monoclonal antibody, polyclonal antibody or antigen) of the present invention can be detectably labeled is by linking the monoclonal antibody, polyclonal antibody, or antigen to an enzyme. This enzyme, in turn, when later exposed to its substrate, will react with the substrate in such a manner as to produce a chemical moiety which can be detected as, for example, by spectrophotometric or fluorometric means.

Examples of enzymes which can be used to detectably label the reagents of the present invention include malate dehydrogenase, staphylococcal nuclease, delta- V- steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase and acetylcholine esterase. The presence of the detectably labeled reagent of the present invention can also be detected by labeling the reagent with a radioactive isotope which can then be determined by such means as the use of a gamma counter or a scintillation counter.

Isotopes which are particularly useful for the purpose of the present invention are ³H,

125j 32p₅ 35_S; 14_Cj 51^ 36^ 57^ 58^ 59^ ^ 75^ It is also possible to detect the binding of the detectably labeled reagent of the present invention by labeling a secondary monoclonal or polyclonal antibody which binds to GPI or an immunogenic portion thereof with a fluorescent compound. When the fluoroscently labeled reagent is exposed to light of the proper wave length, its presence can then be detected due to the fluorescence of the dye. Among the most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

The reagents according to the invention also can be detectably labeled using fluorescent emitting metals such as Eu or others of the lanthanide series. These metals can be attached to the reagent molecule using such metal chelating groups as diethylenetriaminepentaacetic acid (DTP A) or ethylenediaminetetraacetic acid (EDTA), and salts thereof.

The reagents of the present invention also can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent- tagged reagent is then determined by detecting the presence of luminescence that arises during the course of the chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester. Likewise, a bioluminescent compound may be used to label the reagent of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent reagent is determined by detecting the presence of luminescence. Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.

Another technique which may also result in greater sensitivity when used in conjunction with the present invention consists of coupling a monoclonal or polyclonal antibody which recognizes GPI or an immunogenic portion thereof to low molecular weight haptens. The haptens can then be specifically detected by means of a second reaction. For example, it is common to use such haptens as biotin (reacting with avidin) or dinitrophenol, pyridoxal and fluorescamine (reacting with specific anti-hapten antibodies) in this manner.

In general, increases in sensitivity are a development consideration and are achieved by optimization of all reagents, including the concentrations conjugated to reporter systems, adsorbed to solid phase surfaces, specificity of the antibodies (Abs), and affinity of the Abs. These steps are routinely done and evaluated during assay development and are well within the skill of those working in the art.

As is evident to one of ordinary skill, the diagnostic assay of the present 10 invention includes kit forms of such an assay. This kit would include anti-GPI and or antibodies which recognize immunogenic portions of GPI. These antibodies can be polyclonal antibodies (a compositions comprising different antibodies which recognize different GPI epitopes) and/or a monoclonal antibody (Mab) which recognizes only one epitope on GPI. These antibodies can be optionally immobilized, as well as any necessary reagents and equipment to prepare the biological sample for and to conduct analysis, e.g. , preservatives, reaction media such as nontoxic buffers, microtiter plates, micropipettes, etc. The reagent (Abs and/or antigens) can be lyophilized or cryopreserved. As described above, depending on the assay format, the antibodies can be labeled, or the kit can further comprise labeled GPI or immunogenic fragments or analogs thereof containing the relevant epitopes.

The types of immunoassays which can be incorporated in kit form are many. Typical examples of some of the immunoassays which can utilize the antibodies of the invention are radioimmunoassays (MA) and immunometric, or sandwich, immunoassays. "Immunometric assay" or "sandwich immunoassay", includes simultaneous sandwich, forward sandwich and reverse sandwich immunoassays. These terms are well understood by those skilled in the art. Those of skill will also appreciate that the monoclonal antibodies, polyclonal antibodies and/or antigens of the present invention will be useful in other variations and forms of immunoassays, which are presently known or which may be developed in the future. These are intended to be included within the scope of the present invention.

In a forward sandwich immunoassay, a sample is first incubated with a 10 solid phase immunoadsorbent containing monoclonal or polyclonal antibody(ies) against the antigen. Incubation is continued for a period of time sufficient to allow the antigen in the sample to bind to the immobilized antibody in the solid phase. After the first incubation, the solid phase immunoadsorbent is separated from the incubation mixture and washed to remove excess antigen and other interfering substances, such as non-specific binding proteins, which also may be present in the sample. Solid phase immunoadsorbent containing antigen bound to the immobilized antibody is subsequently incubated for a second time with soluble labeled antibody or antibodies. After the second incubation, another wash is performed to remove unbound labeled antibody(ies) from the solid phase immunoadsorbent and removing non-specifically bound labeled antibody(ies). Labeled antibody(ies) bound to the solid phase immunoadsorbent is then detected and the amount of labeled antibody detected serves as a direct measure of the amount of antigen present in the original sample.

Alternatively, labeled antibody which is not associated with the 25 immunoadsorbent complex can also be detected, in which case the measure is in inverse proportion to the amount of antigen present in the sample. Forward sandwich assays are described, for example, in U.S. Pat. Nos. 3,867,517; 4,012,294 and

4,376,110.

Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. The following working examples therefore, specifically point out preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

EXAMPLES

EXAMPLE 1

A method of preparing purified GPI and fragments thereof

After analyzing individually several major GPI-anchored proteins from four different strains of P. falciparum, the GPI anchors on each protein have identical structural features. Therefore, isolation of individual GPI-anchored proteins from P. falciparum, is not routinely necessary.

Large quantities of parasites (30-50 ml of packed parasite-infected red blood cells) are obtained by scaling up cultures. Parasite cultures are labeled with [³H]- glucosamine and mixed with parasites cultured in media without isotopes. After culturing parasites to the mid-schizont stage, parasite-infected erythrocytes are enriched using Percoll gradient centrifugation, as described by Lutz et al, Biochim. Biophvs. Acta 1116: 1-10 (1992). The infected erythrocytes are lysed with 0.015% saponin in PBS, as described by Reese et al, Proc. Nail. Acad. Sci. USA 75: 5665-68 (1978), and passed through a 26 gauge needle to dissociate erythrocyte-membrane fragments still attached to parasites. The parasites are then recovered by centrifugation at 2,500 x g. The parasite pellet is re-suspended in PBS, layered on either 70% Percoll or 5% bovine serum albumin (BSA) in PBS, and centrifuged at 4,500 x g to remove any remaining erythrocyte membranes (Udeinya et al, Bull. W.H.O. 58: 445-48 (1980)). The purified parasites are extracted with chloroform/methanol/water (CMW) (10:10:3, v/v/v) to remove GPI lipids. The organic extract is used for isolation of the free GPI anchors.

The delipidated parasite pellet is extracted with TBS containing 0.5-1.0% 10 CHAPS (dialyzable zwitterionic detergent), and a cocktail of protease inhibitors. If necessary, 6 M guanidine hydrochloride and sonication can be used to solubilize GPI- anchored proteins. The solution is dialyzed exhaustively against 100 mM NHjHCO_. to remove detergent and salts. Some proteins may be precipitated after the removal of guanidine salts and detergents. The solution/suspension is digested with pronase. Complete solubilization of GPI-anchored proteins is not a prerequisite for the release of GPI anchors from the proteins, as GPI anchors can be isolated from gel bands after pronase digestion. Pronase digestion of the delipidated parasite pellet almost quantitatively releases GPI anchors. Exhaustive pronase digestion should leave only the carboxy-20 terminal amino acid linked to the ethanolamine moiety of the GPI anchor. After pronase digestion, the GPI anchor is isolated by extraction with water saturated 1-butanol (Ferguson, "GPI membrane anchors - isolation and analysis," IN GLYCOBIOLOGY: A PRACTICAL APPROACH 350-3 83 (M. Fukuda et al, eds. 1992 a); Ferguson, "Chemical and enzymic analysis of glycosyl-25 phosphatidylinositol anchors," IN LIPID MODIFICATION OF PROTEINS: A PRACTICAL APPROACH 191-231 (N.M. Hooper et al, eds. 1992b); Menon, Meth. Enzymol. 230: 418-42 (1994); Field et al, "Biosynthesis of glycosylphosphatidylinositol membrane anchors," IN LIPID MODIFICATION OF PROTEINS: A PRACTICAL APPROACH 155-190; and Schneider et al, Meth. Enzymol. 250: 614-30 (1995)). The organic phase is back-extracted with water and dried under vacuum. Further purification, if necessary, is achieved by hydrophobic interaction chromatography on octyl-Sepharose as described by McConville et al, J. Biol. Chem. 264: 757-65 (1989).

EXAMPLE 2 Characterization of P. falciparum GPI Intraerythocytic P. falciparum were cultured in vitro and parasites free of red blood cell components were isolated as described above. The free and protein-linked GPI molecules were isolated from the parasites, and purified by preparative high performance thin layer chromatography (HPTLC), as described (Ferguson, 1992a; Ferguson, 1992b; Menon, 1994; Field et al, 1992; Schneider et al, 1995). On HPTLC, the matured, free GPIs (those not anchoring proteins) and protein-linked GPIs migrated as broad major bands. In some HPTLC plates, the parasite GPI separated into two major bands. Carbohydrate compositional analysis of the GPIs purified by HPTLC gave mannose and glucosamine in molar ratios of approximately 4:1. The identity of the GPIs was further evident by their susceptibility to nitrous acid, HF and alkali, which is in agreement with their identity.

The HPTLC-purified GPIs were analyzed for structures by matrix-assisted laser-desorption/ionization mass spectrometry (MALDI-MS). MALDI-MS time of flight mass spectrometry analysis was performed using a Kratos analytical MALKD-4 mass spectrometer equipped with a nitrogen laser at 20-kV accelerating voltage according to Woods et al, Anal. Biochem. 226: 15-25 (1995). Spectra were acquired in negative mode, with a time delayed extraction, and were the average of 50 laser shots. The matrix was a saturated α-cyano-4-hydroxycmnnamic acid in 50% ethanol. The mass analysis reported here are within a mass accuracy of 1 dalton.

The mass spectrum (performed as described above) of free GPIs contained prominent molecular ions (M-H) at m/z 2005.3, 2033.3, and minor ions at m/z 1977.3, 2061.3, and 2089.3 (Fig. 3B). The observed difference of 28 mass units between the consecutive molecular ions suggests that the GPI pool contained mixtures of size isomers containing homologous fatty acids differing by two carbons. The ion at 864.0 is due to the phosphatidylinositol (PI) moiety, with C18:0 and C18:l fatty acids at Cl and C2 of glycerol, respectively, that fragmented with the loss of inositol phosphate to form an ion at m/z 621.1. The ions at m/z 649.0 and 676.2, respectively, correspond 10 diacylglycerol with C20:0 and C22:0 acyl substituents at Cl.

Purified GPIs (0.5-1 g) were treated with bee venom phospholipase A₂ (2400 units/ml) in 100 1 of 100 mM Tris-HCI, 10 mM CaCl₂, pH 7.5, at 37 °C for 18 h, extracted with water-saturated 1-butanol, and dried. Treatment with phospholipase A₂, the enzyme that releases acyl substituents at C2 of glycerol, resulted in the marked loss of (M-H)^" ions and the appearance of ions at m z 1712.9 (1977.3-264.5), 1740.8 (2005.3-264.5), 1768.8 (2033.3-264.5), 1796.8 (2061.3 -264.5), and 1824.8 (2089.3- 264.5) (Fig. 3C). The data establish that the parasite GPIs contain a C18:l acyl substituent at C2 of glycerol. The spectrum also contained an ion at m/z 605.0 that is formed by fragmentation at the C3 of glycerol with the elimination of the hydroxyl oxygen. Together, the data indicate that the major GPI contains C18:0 and C18:l fatty acids at Cl and C2 of glycerol, respectively, and C16:l acyl substituent on inositol; other GPIs contain C14:0, C16:0, C20:0, and C22:0 acyl substituents at Cl.

The mass spectra of the protein-linked GPIs (Fraction I, in Fig. 3A), which contain one amino acid after Pronase digestion, showed prominent (M-H)^" ions at m/z 2092.3 (2005.3 + 87), 2120.4 (2033.3 + 87), and 2148.2 (2061.3 + 87) (Fig. 3D), indicating that these GPIs contain a Ser residue. The predicted amino acid residues that are linked to GPIs in P. falciparum surface proteins, MSP-1 (Miller et al, Molec. Biochem. Parasitol. 59: 1-14, 1993), MSP-2 (Smythe et al, Proc. Natl Acad. Sci. USA 85: 5195, 1988), and MSP-4 (Marshall et al, Infect. Immun. 65: 4460-7, 1997) are Ser, Asn, and Ser, respectively (Fig. 3E). The (M-H)^" ions at m/z 2119.3 (2005.3 + 114) and 2147.3 (2033.3 + 114), not resolved from ions at m/z 2120.4 and 2148.2 respectively, were formed from GPIs with attached Asn residues. However, GPIs with attached Asn are evident from the mass spectrum of Fraction II, which contained (M-H)^" ion at 2175.3 (2061.3 + 114) and a higher proportion of(M-H)^" ion at 2148.2 relative to those at 2092.3 and 2120.4. Thus, these results not only established the amino acids linked to the GPIs of parasite proteins, but also confirmed the identity of the parasite GPIs. Phospholipase A₂ treatment resulted in the loss of (M-H)^" ions with the appearance of new ions at m/z 1827.8 (2092-264.5), 1855.8 (2120.4-264.5), 1883.8 (2148.2-264.5), and 1910.8 (2175.3-264.5) (Fig. 3F), confirming the presence of a C18:l acyl residue at C2 of glycerol.

The acyl substituents in parasite GPIs were also studied by metabolic labeling with [³H]-myristic, [³H]-palmitic, [³H]-palmitoleic, [³H]-stearic, [³H]-oleic, [³H]- linoleic and [³H]-arachidonic acids. Metabolic labeling with radioactive fatty acids was carried out 26 hours after invasion in medium containing 2% human serum and 20 mM glucose. All acids, except arachidonic and linoleic acids, were incorporated into the parasite. Treatment with phospholipase A₂ or HF suggested that myristic, palmitic and palmitoleic acids were incorporated into both the glycerol and inositol residues, stearic acid only to the Cl of glycerol, and oleic acid exclusively to the C2 of glycerol. The parasite is known to synthesize two isomeric C18:l acids (oleic and cis- vaccenic acids) (Holz, Bull. W.H.O. 55: 237, 1977), and therefore, it is likely that the acyl substituent at C2 of glycerol is a mixture of these isomeric acids. The incorporation of palmitoleic acid to glycerol is likely after conversion to cis-vaccenic acid. The incorporation of myristic and palmitic acids to the inositol residue is apparently as a Cl 6:1 acid.

Based on the above results and the previously elucidated carbohydrate core (see Gerold et al, J. Biol. Chem. 269: 2597-606, 1994 and Gowda et al, J. Biol. Chem. 272: 6428, 1997), the GPI structure discussed herein and in Fig. 4 are proposed for the GPIs of P. falciparum. The preferred structure in Fig. 4 is when n = 16. The acyl substituents identified here are different from those previously reported for the P. falciparum GPIs based on metabolic labeling (Gerold et al, Mol. Biochem. Parasitol. 75: 131-43, 1996). The parasite GPIs are different from the GPIs of animals and other microbes thus far characterized with respect to the presence of a C16:l acid on inositol. However, the GPI of a mucin-like glycoprotein from Tiypanosoma cruzi trypomastigotes contains C18:l and C18:2 acyl substituents on C2 glycerol, and the PI moiety elicits a potent proinflammatory cytokine response (Camargo et al, J Immunol. 158:5890-90 1, 1997). In P. falciparum GPI also, the PI moiety is most likely is associated with toxicity and it is the epitope that is recognized by naturally elicited anti-GPI IgGs.

EXAMPLE 3 Isolation of mature GPI anchors not attached to proteins

Since parasites synthesize GPI lipids in excess of that required for protein anchoring, the total lipid extract of the parasite may also provide a good source of mature GPI anchors. [³H]-glucosamine labeled mature GPI anchors can be isolated by thin-layer chromatography, as described by Gerold et al, J. Biol. Chem. 269: 2597-2606 (1994). The hydrophilic glycan head group is isolated by treatment with 25 mM NaOH and Bio-Gel P-4 chromatography. The glycan is analyzed for substituents on the non-reducing-end mannose (Man) residue by preparing the glycan cores before and after treatment with jack bean cc-mannosidase (Ferguson, 1992a; Ferguson, 1992b; Menon, 1994; Field et al, 1992; Schneider et al, 1995). If the non- reducing end Man residue of the mature protein-free GPI anchor also contains substituents as in the case of protein-anchored GPI moieties, then the total lipid fraction of the parasite can serve as a good source for the isolation of GPI anchors for other biological studies.

The total glycolipid fraction of P. falciparum mainly contains GPI with four and three mannose residues (Gerold et al, 1994), with lower proportions of smaller GPI intermediates. Isolation of mature GPI anchors containing all glycan components from the total lipid extract of the parasite is performed by gel filtration using Sephadex LH-60 in 60% aqueous ethanol. This procedure is expected to remove most of the low molecular weight GPI biosynthetic intermediates. For further purification of the mature GPI lipid, hydrophobic interaction chromatography on octyl-Sepharose (McConville et al, 1989) or preparative TLC (Gerold et al, 1994) can be employed. EXAMPLE 4 Mannosamine Inhibition

Mannosamine (ManN), but not glucosamine or galactosamine, has been shown to inhibit parasite growth. Studies were carried out using 1, 2, 5 and 10 mM mannosamine using asynchronous cultures. Parasitemia was measured at 24 h after the addition of mannosamine and was compared with that of a control culture not receiving mannosamine.

EXAMPLE 5 Preparation of monoclonal antibodies against P. falciparum GPI anchors Production of hybridomas will be as described in Harlow et al, ANTIBODIES:

A LABORATORY MANUAL (1988); Campbell, "Monoclonal Antibody Technology: The Production and Characterization of Rodent and Human Hybridomas," IN LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY 13: 1-242 (R.H. Burdon et al, eds. (1987)); Beverly, 15 Monoclonal Antibodies: Methods Hematology 13: 1-269 (1986); and Coligan, Current Protocols in Immunology (1992). Specifically, BALB/c mice are immunized by s.c. or i.v. injection with various amounts (10-50 g) of purified GPI anchors (with TiterMax as adjuvant) isolated from pronase-treated P. falciparum proteins, as described above. After two weeks, the animals are boosted with similar amounts of antigen. Eight to ten days later, the animals are screened (by ELISA) for anti-GPI antibodies. Those animals with highest response are administered a final i.v. boost, sacrificed 3 days later, and their spleens recovered. Spleen cells are prepared, fused with mice myeloma cells and hybrid cells selected in HAT (hypoxanthine/aminopterine/thymidine) medium. Anti-GPI antibody producing hybridomas are selected first by limiting dilution methods, followed by cloning. The clones are further screened for epitope specificity using the previously prepared GPI anchors with defined structural features.

Screening Methods. Antibody titers and screening of hybridoma culture supematants are tested using ELISA. Purified P. falciparum GPI anchors are coated onto wells of 96-well microtiter plates and non-specific binding sites blocked by 2% bovine serum albumin (BSA). Hybridoma culture supernatants (100 1 ) will be added in duplicate and added at room temperature. After extensive washing with TBS, the bound antibodies are measured by using alkaline phosphate or horseradish peroxidase conjugated goat-anti-mouse immunoglobulin (Ig) and the NBT BCIP chromogenic substrate. The determination of the class (lgM or IgG) and subclass (IgGi, JgG2a, IgG2b, etc.) of monoclonal antibodies (isotyping) will be done by ELISA using antibody capture on anti-Ig antibodies, as described in Harlow, 1988; Campbell, 1987; Beverly, 1986; and Coligan et al, 1992.

Mapping of epitope specificity of P. falciparum anti-GPI MAbs. To determine the structural features of parasite GPI anchors that are specifically recognized by monoclonal antibodies, the ELISA procedure outlined above will be used (Harlow, 1988; Campbell, 1987; Beverly, 1986; and Coligan et α/.,1992). The GPI anchors are selectively modified to remove certain elements of their structures and then analyzed by ELISA. These epitope-defmed MAbs are then used in confirming the structural specificity of the parasite GPI anchors in the activation of vascular endothelial cells. The antibodies are pre-incubated with various GPI anchors (e.g., compounds A to F, as discussed in Example 6, below) to assess whether the epitope-specific MAb specifically neutralizes the activity of the GPI anchor, i.e., the up-regulation of ICAM-1 expression and cytoadherence of parasite-infected erythrocytes to activated-endothelial cells.

EXAMPLE 6 Testing and Preparation of P. falciparum GPI anchors not containing unusual substituents

Preliminary data suggests that only 50-60% of the terminal mannose residues of the P. falciparum protein GPI anchors are substituted. The GPI species lacking the substituent contain a terminal 1 ,2-linked mannose that is expected to be preferentially recognized by ConA, a lectin that has high affinity for α-mannosyl residues (Merkle et al, Enzymol. 138: 232-59, 1987). Therefore, ConA-affinity chromatography is used to isolate a population of GPI structures lacking the substituents. The GPI anchors isolated after pronase digestion are loaded onto a ConA-Sepharose column in-the presence of a non-ionic detergent. After washing with buffer lacking detergent, the bound GPI anchors are eluted with buffer containing methyl α-mannoside, and if necessary, 20-30%) 1-propanol. The GPI anchors will be isolated from the eluent by butanol extraction.

GPI structures containing only three mannose residues are produced by treatment of the above product with jack bean a-mannosidase. The following chart illustrates this isolation strategy:

(A) R-(X-Manαl-2)6Manαl-2Manαl-6Manαl-4GlcN-PI

+

(B) R-(Manα 1 -2)6Manα 1 -2Manα 1 -6Manα 1 -4GlcN-PI

I I . ConA-Sepharose

I I

R-(X-Manα 1 -2)6Manα 1 -2Manα 1 -6Man<x 1 -4GlcN-PI |

(A, not bound or weakly bound to ConA) |

I R-(X-Man<xl-2)6Manαl-2Manαl-6Manαl-4GlcN-PI

(B, bound to ConA) |

I Jack bean α-mannosidase treatment

I (C) R-6Manαl-2Manαl-6Manαl-4G_cN-PI

(A) HF/HN0₂ NaBH₄ >Mancc 1 -2Manα 1 -2Manc 1 -6Manα 1 -4 AHM

(glycan core, D)

(A) mild alkali >R-(Manα 1 -2)6Manα 1 -2Manα 1 -6Manα 1 -4GlcN-inositol-P-glycerol

(hydrophillic head group, fatty acid free, E)

(A) HN0₂ >R-(Manα 1 -2)6Manα 1 -2Manα 1 -6Manα 1 -4(2,5 -anhydroMan) (hydrophillic head group, devoid of Pi moiety, F)

The GPI anchor structures (compounds A to F) listed above can then be tested for up- regulation of adhesion molecules in human umbilical vein endothelial cells (HUVEC) and for cytoadherence. Primary cultures of HUVEC (Clonetics, Corp., San Diego, CA) are grown 25 in medium 199 (Gibco) containing 10-20%) fetal calf serum, 50 g/ml endothelial cell growth supplement, and 100 g/ml heparin (Mannoriet al., Cancer Res. 55: 4425- 4431(1995)). For subculturing, cells are detached with trypsin and split 1:3. To measure up-regulation of adhesion molecules by any of compounds A to F, cells are cultured in 12- or 24-well plates. The measurements are performed between the third and fifth passages, as discussed below.

Measurement of cell adhesion molecule expression in HUVEC. Previous studies suggest that the P. falciparum GPI anchors up-regulate the expression of ICAM- 1 , VCAM- 1 and E-selectin in IHUVEC (Schofield et al, 1996). Among these, induction of ICAM-1 was more pronounced, in that a significant level of ICAM-I induction was observed even at 0.1 M of parasite GPI. ICAM-I levels will be measured to determine which structural feature of the glycan moiety contributes most to up-regulation of adhesion molecules. HUVECs cultured in 12- or 24-well plates are treated with 1, 5 and 10 M various GPI anchor fractions (e.g., A to F) isolated as discussed above, for 24 hours. Cells are harvested in either by scraping and suspending in PBS or culture medium containing 1% bovine serum albumin at 4 °C. The level of ICAM-1 expression is measured by FACS analysis after fluorescein- staining of cells with anti-ICAM-1 MAb (anti-CD54; Calbiochem, La Jolla, CA) as the first antibody, and fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin, as the second antibody. Cells not treated with GPI anchors will also be stained in parallel and analyzed by FACS for the presence of constitutively expressed ICAM-1 (Schofield et al, 1996). Cells will be counter-stained with propidium (0.5 g/ml) to gate-out non-viable cells. Cells treated with an irrelevant MAb (same isotype as that of anti-CD54) are similarly analyzed as controls (background fluorescence); about 10,000 cells are analyzed per sample. As an alternative to using two antibodies (primary and secondary) for staining, FITC- conjugated mouse anti-ICAM-1 (Calbiochem) can be employed for direct staining.

Epitope-specific monoclonal antibodies (described above) are used to confirm the structural features of the parasite GPI anchors that up-regulate the expression of ICAM-1. For these experiments, the GPI fractions are incubated with various MAbs, and then added to the HUVEC culture and ICAM-1 measured, as described above.

Adherence of P. falciparum-infected erythrocyte to GPI-treated HUVEC. HUVECs are grown on glass cover slips in 24-well plates. The confluent cells are treated with 0.1, 1.0 and 10 M GPI anchor fractions, obtained as previously described in Schofield et al, 1996. The cells are washed with complete medium and then incubated with P. falciparum-infected erythrocytes in complete medium at 37 °C for 1 hour. After removing the supernatent, cell are washed with complete medium, fixed with 2% glutaraldehyde, and stained with Giemsa. The number of bound parasite-infected erythrocytes is counted by light microscopy (Schofield et al, 1996). Parallel studies using epitope specific MAbs to prevent cytoadherence can also be performed.

EXAMPLE 7 Screening for Antibodies Which Block Merozoite Invasion of Erythrocytes

Plasma membranes of merozoites undergo extensive modification during parasite entry into red blood cells (Gilles et al. (eds.), BRUCE-CHWATT'S ESSENTIAL MALARIOLOGY 1-340 (1993)). The GPI anchors may play a role in entry of the merozoites into the red blood cell. Accordingly, antibodies which bind to GPI anchors can be screened for the ability to block entry of the merozoites into red blood cells. Parasites are cultured in complete medium and synchronized to the ring stage with sorbitol (Lambros et al, J. Parasitol. 65: 449-462 (1994)). Various amounts (1, 10 and 100 g/ml medium) of monoclonal antibodies against GPI anchors or epitopes thereon are added to the parasite culture at the schizont stage and subsequent merozoite invasion measured by determining parasitemia several hours after the expected completion of invasion. The parasitemia of parasite cultures not treated with the monoclonal antibodies or treated with a control monoclonal antibody are measured in parallel. EXAMPLE 8 Mannosamine as an Inhibitor of P. falcin arum Growth and Development

The ability of mannosamine to inhibit P. falciparum growth and development is determined as follows. Parasite cultures with -12% parasitemia are synchronized to the ring-stage by treating with 5% sorbitol ((Lambros et al, 1994), and then washed and suspended in complete medium. After 4 to 5 hr incubation in complete medium containing 10% human serum, parasitemia is adjusted to 4-5% (3% hemocrit) by the addition of fresh erythrocytes. The cultures are treated with various concentrations of mannosamine (ManN) (0.1 to 10 mM) at different developmental stages (e.g., rings, early trophozoites, trophozoites, early schizonts and late schizonts). The growth and development of the parasites are monitored by counting the infected erythrocytes in Giemsa-stained thin blood smears under light microscopy. Parasite cultures not treated with ManN are used as controls. These studies establish the optimal concentration of ManN for maximum P. falciparum toxicity and the developmental stage at which ManN toxicity is the most effective. This experiment can be employed to test ManN inhibiting activity in other species of Plasmodium.

Effect of ManN on P. falciparum GPI anchor and protein synthesis and attachment of GPI to proteins. Although unlikely, ManN may affect protein synthesis at higher concentrations (5-10 mM). To establish that the observed toxicity of ManN and its impact on GPI biosynthesis, metabolic labeling of parasites is performed at different stages of erythrocytic development using [³⁵S]-methionine in the presence and absence of various amounts of ManN (0.1 to 10 mM). Percoll- enriched parasite-infected erythrocyte cell lysates are analyzed by SDS-PAGE fluorography or phosphoimager quantitation (Laemmli, Nature 227: 680-685(1970); Chamberlain, Anal. Biochem. 98:132-135 (1979)). A significant reduction in the amount of radiolabeled proteins is observed if ManN impairs general protein synthesis.

GPI biosynthetic products formed in the presence of ManN are analyzed as a function of ManN concentration and parasite growth stage. Parasite cultures at rings, trophozoites, schizonts stages are treated with various concentrations of ManN for 1 to 2 hours (as described in Gowda et al, J. Biol. Chem. 272: 6428-6439 (1997)) and then metabolically labeled using either [³H]-mannose, [³H]-røvo-inositol or [³H]- ethanolamine for 2 to 4 hours. Control parasites (not treated with ManN) are similarly labeled with radioactive sugar precursors. After metabolic labeling, the parasite-infected erythrocytes are enriched with an equal volume of water-saturated 1- butanol (Ferguson, 1992a; Ferguson, 1992b, Menon, 1994; Field et al, 1992; and Schneider et al, 1995). The 1-butanol phase contains all the GPI lipids and their biosynthetic intermediates. These are analyzed by HPTLC (Ferguson, 1992a; Ferguson, 1992b; Menon, 1994; Field et al, 1992; and Schneider et al, 1995). Although erythrocytes do contain GPI-anchored proteins, they do not carry out any biosynthetic activity and will not radiolabel such structures.

Parallel to lipid analysis, the GPI-anchored proteins from parasite cultures 25 are radiolabeled with [³H]-glucosamine and analyzed by SDS-PAGE fluorography. Parasite proteins are extracted from the delipidated, parasite-infected erythrocyte pellet with TBS/1% SDS by sonication and analyzed by SDS-PAGE fluorography. Controls and ManN-treated cultures (from equal number of infected erythrocytes) are processed in parallel. Parasites not treated with ManN should provide a GPJ- anchored protein similar to that depicted in Figure 1. Parasites treated with ManN are expected to contain significantly less or no radiolabeled GPI-anchored proteins.

The lack of [³H]-glucosamine-labeled proteins in the parasites treated with ManN may not necessarily indicate the lack of formation of GPI-anchored proteins. It is possible that such proteins are formed utilizing a pre-existing GPI anchor pool that is not radiolabeled. To ascertain whether this is occurring, parasites are labeled with [³⁵5]-methionine in the presence and absence of effective concentrations of ManN (determined from the above experiments; i.e., sufficient to cause maximal inhibition of label incoφoration into GPI) (Field et al, J. Biol. Chem. 268: 19570-9077 (1993)). MSP-1, a GPI-anchored protein, is analyzed after immunoprecipitation from control and ManN-treated cell lysates (from equal number of parasite-infected erythrocytes). Since newly synthesized proteins attach to GPI anchors within a minute after their synthesis in the endoplasmic reticulum, the formation of [ S]-methionine labeled MSP-I suggests two possible situations: (1) The presence of a large GPI pool that is available for protein attachment; and (2) mature GPI anchors that are formed even at a slower rate would be sufficient for newly synthesized protein to be GPI anchored. However, the toxicity of ManN to P. falciparum suggests that the sugar is completely inhibiting GPI anchor synthesis.

EXAMPLE 9 Benzyl- or Phenyl-α-2-amino-2-deoxy-glucosaminides as P. falciparum GPI Anchor Inhibitors

This experiment tests the inhibitory activity of benzyl- or phenyl-α-2-amino-2- deoxy-glucosaminides against P. falciparum GPI anchor synthesis. It is established that aryl glycosides can serve as artificial acceptors for glycosyltransferases (Huang et al, Oncol. Res. 4: 507-5015 (1992); Sarkar et al, Proc. Nat'l. Acad. Sci. 92: 3323- 3327 (1995); Lugemwa et al, J. Biol. Chem. 266: 6674-6687 (1991); Salimath et al, J. Biol. Chem. 270: 9164-9178 (1995)), which inhibit specific types of protein glycosylation. These glycosides can enter the Golgi apparatus of the cells, where they compete with natural glycosylation acceptor sites on proteins resulting in the elaboration and secretion of free oligosaccharide chains built on these glycosides, inhibiting protein glycosylation. Another compound, aryl-2-amino-2-deoxy- glycosaminide may also compete with GPI anchor synthesis, specifically with GlcN- Pi, the first intermediate in the GPI anchor biosynthetic pathway. This compound (as well as other similar compounds) can be tested like ManN, discussed above, to selectively and effectively inhibit P. falciparum GPI biosynthesis. The following materials were utilized in Examples 10-13 which follow. Materials and Methods

Reagents. RPMI 1640, DME culture medium, and cell culture reagents were from Life Technologies. Human blood and serum were purchased from Interstate Blood Bank (Memphis, TN). Gelatin, bee venom phospholipase A₂ (1800 units/mg), standard phospholipids, and saponin were from Sigma. Silica Gel 60 HPTLC plates were from either EM Science or Whatman. Pronase was purchased from Calbiochem. Aspergillus saitoi -mannosidase (400milliunits/mg), and jack bean -mannosidase (30 units/mg), were from Oxford Glycosystems. Poly(isobutyl methacrylate) was procured from Polysciences. HRP-conjugated goat anti-human IgG (H+L chains) and ABTS reagent were from Kirkegaard & Perry Laboratories (Gaithersburg, MD). Microtiter plates were from Dynex Technologies, Chantilly, VA. [6-³H]GlcN (23 Ci/mmol), [³H]arachidonic acid (202 Ci/mmol), [³H]myristic acid (55 Ci/mmol), and [³H]oleic acid (8 Ci/mmol), were from Amersham-Pharmacia Biotech. [³H]linoleic acid (60 Ci/mmol), [³H]palmitic acid (60 Ci/mmol), [³H]palmitoleic acid (60 Ci/mmol), and [³H]stearic acid (60 Ci/mmol) were procured from American Radiolabeled Chemicals. ¹²⁵I-labeled goat anti-human IgG (8.66 C i/g), and En³Hance were from Dupont-NEN. Murine macrophages (J774A.1) were obtained from ATCC. HPLC grade solvents were used throughout the study. Cell culture and isolation of parasites. Intraerythrocytic P. falciparum (FCR-

3 strain) was cultured in RPMI 1640 medium using human O-type erythrocytes and 10% O-positive human serum at 3 to 4% hematocrit [Naik, R.S. et al., J. Biol. Chem. (in press, 2000); Gowda, D.C. et al., J. Biol Chem. 272:6428-6439 (1997)] . Cultures were synchronized with 5% sorbitol [Lambros, C. et al., J. Parasitol. 65:418-420 (1979)]. Cultures were routinely tested for mycoplasma [Rowe, J.A. et al, Mol Biochem. Parasitol. 92:177-180 (1998)], and only contamination-free cultures used.

Cultures with 20-30% parasitemia were harvested at mid-schizont stage. Packed erythrocytes (20 ml) were suspended in 180 ml of 0.65% gelatin in culture medium and incubated at 37 °C for 30 min [Jensen, J.B., Am. J. Trop. Med. Hyg. 27:1274-1276 (1978)]. The enriched infected erythrocytes (70-80%) in the supernatant were recovered, and lysed with 0.015% saponin in Trager's buffer [Trager, W., Exp. Parasitol. 8:265-273 (1959)]. The suspension was passed through a 26-gauge needle to solubilize the erythrocyte debris, and centrifuged. The parasites were suspended in the above buffer, layered on 5% BSA, and centrifuged [Udeinya, I.J. et al., Bull. W.H.O. 58:445-448 (1980)]. The packed parasites (~4 ml) were washed with the buffer and stored at -80 °C until used.

Metabolic labeling of the GPIs. Parasites were radiolabeled in medium containing 5 mM glucose and [³H]GlcN (50 Ci/ml) [Gowda, D.C. et al,J. Biol. Chem. 272:6428-6439 (1997)]. Labeling with [³H]fatty acids (50 C i/ml) was performed in medium containing 2% serum and 20 mM glucose.

Isolation of GPIs. All procedures were carried out using acid-washed, siliconized glasswares to prevent surface adsorption of GPIs, high quality solvents, and sterile water and buffers. Parasites (10 ml) were lyophilized, and extracted 3 times with 50 ml of chloroform/methanol (2:1, v/v) to remove nonglycosylated lipids. The pellet was extracted 5 times with 50 ml of chloroform/methanol/water (CMW, 10:10:3, v/v/v), dried and then partitioned between water and water-saturated 1- butanol. The organic layer was dried and the residue was extracted with 80% aqueous 1-propanol. Finally, GPIs were purified by HPLC and HPTLC. To isolate the protein-linked GPIs, the delipidated parasite pellet was digested with pronase (50 units/ml) in 50 ml of 100 mM NH₄HCO₃, 1 mM CaCl₂, pH 8.0, at 37 °C for 24 h. The released GPIs were extracted with water-saturated 1-butanol, washed with water, dried, and purified by HPLC. GPIs (2 g) were hydrolyzed with 2.5 M TFA at 100 °C for 5 h, and analyzed for mannose content by HPLC [Hardy, M.R. et al., Methods Enzymol. 230:208-225 (1994)]. Control erythrocyte membrane debris, saponin-lysate, and ghosts, all were similarly extracted and fractionated.

HPLC purification of GPIs. The GPIs (-10 g plus 400,000 cpm of [³H]GlcN-labeled GPIs) was chromatographed on a C₄ reversed phase Supelcosil LC- 304 HPLC column (4.6 X 250 mm, 5 m particle size,Supelco, Bellefonte, PA) using a linear gradient of 20 to 60% aqueous 1-propanol containing 0.1% TFA over a period of 80 min, and hold for 30 min at a flow rate of 0.5 ml/mm (37). Fractions (1.0 ml) were collected and elution of GPIs monitored by measuring radioactivity. Aliquots (0.5 1) were also assayed by ELISA with Kenyan adult sera. Glycolipids extracted from control erythrocyte membrane debris, total lysate, and ghosts were similarly chromatographed and fractions were analyzed for reactivity with Kenyan sera.

Purification and analysis of GPIs by HPTLC. The HPLC-purified GPIs (5 g) were applied onto 10 X 10 cm plates as continuous streaks. Parallel spots with [³H]GlcN-labeled GPIs (50,000 cpm) were used for monitoring GPI bands by flurography using En³Hancer (18,19). The plates were developed with CMW (10:10:2.5, v/v/v). The GPIs from plates were extracted with CMW (10:10:3, v/v/v), dried, dissolved water-saturated 1-butanol, and washed with water.

In separate experiments, HPTLC plates were scraped (0.5-cm width fractions), GPIs extracted, and analyzed by ELISA using Kenyan adult sera. Glycolipids from control erythrocyte membrane debris, total lysate, and ghosts were similarly analyzed.

Mass spectrometry. Matrix-assisted laser-desorption/ionization time of flight mass spectrometry analysis was performed using a Kratos analytical MALDI-4 mass spectrometer equipped with a nitrogen laser at 20-kV accelerating voltage [Woods,

A.S. et al., Anal. Biochem. 226:15-25 (1995)]. Spectra were acquired with a time- delayed extraction, and were the average of 50 laser shots. The matrix was saturated with -cyano-4-hydroxycinnamic acid in 50% ethanol. The mass accuracy is within 1 Dalton.

7NE- induction by GPIs. Murine macrophages (1.5X10⁶cells/well in 96- well plates) in DMΕM and 10% fetal bovine serum were stimulated with 0.03-0.5 M of HPTLC-purified free GPIs (Fig. IB lane 2). TΝF- in the culture supematants was measured using a quantitative sandwich ΕLISA using the TΝF- measurement kit (R and D System, Minneapolis, MΝ). Experiments were performed in triplicate and with three separate batches of GPI preparations. IgG-specific ELISA. HPLC-purified GPIs, dissolved in methanol (0.25 to 32 ng/well), were coated onto 96-well microtiter plates. After evaporation of methanol at 37 °C, the wells were blocked with 0.5% casein in 50 mM Tris-buffered saline, pH 7.4 (TBS-casein), and incubated with serially diluted sera (1:100 to 1:64,000) in TBS- casein containing 0.05% Tween-20. Initially, randomly selected 20 Kenyan and 2 USA adult sera were assayed. The bound antibodies were measured by HRP- conjugated goat anti-human IgG and ABTS substrate. All other sera were analyzed at 1:200 or 1:400 dilution by coating GPIs at 0.5 to 2 ng/well.

Competitive inhibition ELISA. Sera were 1:200 diluted in TBS-casein containing 0.05% Tween-20, and incubated at room temperature for 30 min with various concentrations of Pis, PG, CL or GPI, and then ELISA was performed as described above.

TLC-immunoblotting. GPIs (HPLC not purified, 100 ng each) were chromatographed on HPTLC plates. The plates were soaked in 0.1% poly(isobutyl methacrylate) in hexane, dried, and incubated for 2 h at room temperature with TBS containing 1% BSA, and then for 2 h in l:100-diluted sera [Magnani, J.L. et al., Methods Enzymol. 138:195-207 (1987)]. The plates were washed with cold phosphate-buffered saline, incubated with ¹²⁵I-labeled goat anti-human IgG (5 C i/ml) for 1 h, washed, dried, and exposed to X-ray film. Nitrous acid treatment. The HPLC-purified GPIs (20 g) suspended in 150 1 of 0.2 M NaOAc, pH 3.8, were treated with 150 1 1M NaNO₂ at room temperature for 24 h (40). The released PI moieties were extracted with water-saturated 1 - butanol. The glycan moieties were recovered by chromatography on a Bio-Gel P-4 column (1 X 90 cm) in 100 mM pyridine, 100 mM HOAc, pH 5.2. These were used to assess seroreactivity.

Treatment with HF. The HPLC-purified GPIs (2 g p lus 500,000 cpm of [³H]GlcN-labeled GPIs) were treated with 50% aqueous HF (50 1) in an ice bath for 48 h (40). The reaction mixture was neutralized with LiOH and extracted with water- saturated 1-butanol. The organic layer was washed with water, dried, and the carbohydrate moiety was purified by HPLC.

Treatment with mannosidase. The [³H]GlcN-labeled GPI glycan cores (10,000 cpm) with digested with jack bean -mannosidase, products were deionized with AG 50W-X16 (H*), and lyophilized [Gowda, D.C. et al., J. Biol Chem. 272:6428-6439 (1997)]. The glycan cores (5,000 cpm) were also digested with Aspergillus saitoi -mannosidase (0.5 milliunits/ml), deionized, and lyophilized [Gowda, D.C. et al., J. Biol Chem. 272:6428-6439 (1997)].

Treatment with phospholipase A₂. HPLC-purified GPIs (2 g) in 100 1 0 f 100 mM Tris-HCI, 10 mM CaCl₂, pH 7.5, were treated with bee venom phospholipase A₂ (2400 units/ml) at 37 °C for 18 h. The digest was extracted with water-saturated 1- butanol, and purified by HPLC; the retention time of lyso-GVls is 75.5 mm.

Analysis of cohort sera for Anti-GPI antibodies. Sera were from subjects of a longitudinal malaria project performed in a holoendemic, rural region of Western Kenya, within the context of the Asembo Bay Cohort Project (ABCP) [Branch, O.H. et al., J. Infect. Dis. 181:1746-1752 (2000)]. Studies in the subjects' households show that from March to May the entomological inoculation rate (EIR) is ~30 infected bites per person in a month, whereas during the drier months the EIR is -4. Pregnant mothers were identified and, after written consent, they and their delivered infants and older children living in the same household were enrolled. The mothers and children were followed every two weeks for -4 years, and recorded clinical parameters including axillary temperature and illness over the past week. Blood samples were taken monthly and parasite and hemoglobin density were measured. >90% of detected parasites contained only P. falciparum species. Sera of one hundred 7-8 year old children and 50 non-pregnant adults were analyzed at two time points, during February- August 1993, spaced 1-3 months apart. For data analysis, we selected two time points from the young children, in the same manner as the older individuals, when they were approximately 0.5, 1.5, 2.5, and 3.5 years old. The study was approved by the Centers for Disease Control and Prevention, and Kenyan Institutional Review Boards.

Analysis of the relationship between serum anti-GPI antibodies with resistance to malaria pathogenesis. Analysis for correlation of anti-GPI antibody response with either hemoglobin or temperature as the dependent variable was done with the general linear model, Generalized Estimating Equations [Zeger, S.L. et al., Biometrics 42:121-130 (1986)]. The variables considered in the analyses were (i) anti-GPI antibody responder category (positive, intermittent, or negative), (ii) parasite density, log₁₀ (n+1/ 1), (iii) age, and (iv) anti-malaria drug treatment. We controlled for the repeated measures on each patient. The estimated effect (EE) of positive and negative antibody responder category are given relative to that of the intermittent antibody responder category.

EXAMPLE 10

Isolation and purification of GPIs from intraerythrocytic P. falciparum.

Contamination with glycolipids from host erythrocytes and mycoplasma is a major concern in isolating GPIs from intraerythrocytic P. falciparum; we used rigorous protocols. First, parasite cultures were routinely tested for mycoplasma, and parasite- infected erythrocytes were enriched from cultures with 25-30% to 70-80% parasitemia to minimize erythrocyte components. Second, the parasites were released from infected erythrocytes by mild saponin lysis, washed, and purified by density centrifαgation to remove erythrocyte membrane debris; metabolically [³H]GlcN- labeled parasite cultures revealed that this procedure did not release parasite GPIs. These procedures eliminated all the erythrocyte components. Third, the purified parasites were differentially extracted to remove most nonglycosylated lipids, and the extract containing the free GPIs (not linked to proteins) was subjected to solvent fractionation to eliminate soluble hemozoin and other colored components. Finally, the GPIs were purified by successive fractionation using HPLC and HPTLC. The protein-linked GPIs were isolated by a similar protocol after exhaustive digestion of the delipidated parasites with pronase. The yields of free and protein-linked GPIs were 100-120 g and 25-30 g per 10 ml of packed purified parasites, respectively, based on the mannose content.

The parasite GPIs were purified by HPLC using a C₄ reversed phase column, which separated matured GPIs from GPI intermediates (Fig. 4A). The elution of GPIs was monitored by radioactivity, which overlapped with the reactivity of the GPIs to human sera containing anti-GPI antibodies (Fig. 4A). The compounds isolated from the extracts of erythrocyte debris, lysates, and ghosts were fractionated in parallel with the purification of parasite GPIs by HPLC (Fig. 4 A) and HPTLC, and analyzed as controls. These did not react with Kenyan adult sera (Fig. 4A), suggesting that the activity is specific to parasite GPIs.

The GPIs were further purified by HPTLC; they migrated as broad major bands and in some instances separated into two major bands (Fig. IB); these were differentially pooled for mass spectrometry (Fraction I and II in lane 4). The heterogeneity of GPIs is due to the variation in the fatty acid composition of individual GPIs (see below). The purified matured GPIs (Fig. 5B, lanes 2 and 4) contained mannose and glucosamine in molar ratios of -4:1, and were susceptible to nitrous acid, HF and alkali (not shown). The identity of the GPIs was established by structure determination. Structural analysis of P. falciparum GPIs. The structures of the purified P. falciparum GPIs were determined by specific chemical and enzymatic degradation studies using [³H]GlcN- and [³H]fatty acid-labeled GPIs and by mass spectrometry.

The [³H]GlcN-labeled GPIs were characterized by subjecting them to various degradative procedures. Treatment with jack bean -ma nnosidase quantitatively converted the matured [³H]GlcN-labeled GPIs into species with three mannose residues similar to recently reported results on GPI biosynthesis [Naik, R.S. et al., J. Biol. Chem. (in press, 2000)], suggesting that the distal fourth mannose residue does not contain any substituent. The [³H]GlcN-labeled GPIs were dephosphorylated with aqueous HF, and then deaminated and reduced with NaBFL. The neutral glycan core, thus obtained, on HPTLC analysis migrated as a single band with an R_f value identical to Mar^-AHM previously characterized. Treatment of the glycan core with A. saitoi -mannosidase shifted the R_f value to that of authentic Man₂-AHM, and the product of jack bean -mannosidase digestion co-migrated with authentic AHM (not shown). These results are consistent with the previously established structure, Man l -2Man -2Man l-6Man l -4GlcN, for the glycan core of P. falciparum GPIs. The mass spectrum of the purified matured GPIs (upper half portion of the HPTLC band in Fig. 1 B, lane 2) contained prominent molecular ions (M-H)^" at m/z 2006.3, 2034.3, and minor ions at m/z 1978.3, 2062.3, and 2090.3 (Fig. 5A). The observed difference of 28 mass units (in some fractions 26 units) between the consecutive molecular ions suggests that the GPI pool contained mixtures of size isomers containing homologous fatty acids differing by two carbons. The proportions of the GPI species varied considerably depending on the regions of HPTLC band analyzed (compare Fig. 5A with Fig. 5B), suggesting marked heterogeneity with respect to acyl substitutents. Mass spectrometry of the control materials did not show molecular ions comparable to the parasite GPIs (not shown). These results show that the purified compounds are GPIs of the parasite, and not from erythrocytes.

Treatment of free GPIs with phospholipase A₂ resulted in the marked loss of (M-H)^" ions of intact GPIs and the appearance of a new set of ions each with 264.5 mass units lower (Fig. 5B), indicating that the GPIs contain a C 18:1 acyl substituent at the sn-2 position. The spectra of some GPI fractions treated with phospholipase A₂ also contained significant levels of ions with 262.5 mass units lower than the parent ions, suggesting the presence of GPIs with a C 18:2 acid at sn-2. These data indicate that the GPIs contain C18: 1 (major) and C18:2 (minor) fatty acids at sn-2. As previously reported [Gerold, P. et al., Mol. Biochem. Parasitol. 75:131-

143 (1996)], the GPIs were sensitive to GPI-specific phospholipase D, but completely resistant to Pl-phospholipase C (data not shown), suggesting the presence of an acyl substituent on C-2 of inositol. To identify this substituent, the GPIs were treated with HF and the released inositol-acylated carbohydrate moiety purified by HPLC as a single symmetrical peak with retention time of 47 min. Positive ion-mode mass spectrum showed (M+Na⁺) ions at m/z 1222.4 and 1252.7 (Fig. 5C), which were assigned to MarL_t-GlcN-inositol with myristoyl and palmitoyl substitution, respectively, on inositol. The signals at m/z 1228.7 and 1266.8 represent (M+H*) and (M+K⁺) ions of the carbohydrate with palmitoyl substitution. Of several mass spectra recorded, some (not shown) showed a reasonable signal at m/z 1200.4, which was assigned to (M+H⁺) ion of the carbohydrate with myristoyl substitution. A significant portion of the HF-released glycan moieties retained a phosphate ester group as indicated by the negative ion mode spectrum, which showed molecular ions at m/z 1280.0 and 1308.1 (Fig. 5D). The phosphate ester group that survived HF treatment is likely the one linked to inositol, because the adjacent protonated GlcN residue will render the hydrolysis of the phosphate ester bond kinetically slower. These results established that, in the inositol moiety, 70% and 30% of the GPIs are substituted with palmitate_and myristate, respectively. Together, the above data suggest that sn-\ is substituted with a range of C 14:0 to C22:0 fatty acids.

The mass spectra of the protein-linked GPIs (Fraction I in Fig. 4B) that contain one amino acid after pronase digestion, showed prominent molecular ions that are 87 mass units higher than those of free GPIs (compare Figs. 5E and 5F with 2 A), indicating that these GPIs contain a Ser residue. GPIs with attached Asn were also evident from (M-H)^" ions at m/z 2120.3, 2148.3, and 2176.3 in the mass spectrum of Fraction II (not shown). This agrees with the predicted amino acids in GPI-anchored surface proteins [Miller, L.H. et al., Mol. Biochem Parasitol. 59:1-14 (1993); Smythe, J.A. et al., Proc. Natl Acad. Sci. USA 85:5195-5199 (1988); Marshall, V.M. et al., Infect. Immun. 65:4460-4467 (1997)], MSP-1, MSP-2, and MSP-4 (Fig. 2 G). Phospholipase A2 treatment caused reduction in m/z of each of the molecular ions, by 264.5 mass units (Fig. 5F), confirming the presence of predominantly a C18:l acyl residue at sn-2. Since the parasite can synthesize two isomeric C18:l fatty acids (oleate and cz^'s-vaccenate) [Holz, G.G. Jr., Bull W.H.O. 55:237-248 (1977)], the acyl substituent at sn-2 may be a mixture of these isomers. The acyl substituents in parasite GPIs were also studied by metabolic labeling with [³H]myristic, [³H]palmitic, [³H]palmitoleic, [³H]stearic, [³H]oleic, [³H]linoleic and [³H]arachidonic acids. All except arachidonate were incoφorated into the parasite (Fig. 6 and data not shown). Treatment of the GPIs labeled with [³H]myristic and [³H]palmitic acids with phospholipase A₂ showed the presence of incoφorated label only in the /vso-GPIs; the released fatty acids were not radioactive (Fig. 6). On treatment with HF, the radioactivity was found to be present on both the diacylglycerol and inositol-carbohydrate moieties (not shown). In the case of [³H]stearic acid, the radioactivity was incoφorated only to the diacylglycerol moiety (not shown). These results agree with the presence of saturated fatty acids at sn-l and at C-2 of inositol, but not at sn-2, established by mass spectrometry. Treatment of [³H]palmitoleic, [³H]oleic, and [³H]linoleic acid-labeled GPIs with phospholipase A₂, released more than 90% of the incoφorated activity as free acid; the / so-GPIs contained negligible radioactivity (Fig. 6). Treatment with HF showed that most of the incoφorated radioactivity was on the diacylglycerol moiety and the inositol- carbohydrate moiety was not radioactive (not shown). These results agree with the presence of unsaturated fatty acyl residues at sn-2 indicated mass spectrometry. The incoφoration of [³H]palmitoleic acid to sn-2 could be after its conversion to cis- vaccenic acid, since the parasite has capability to synthesize -vaccenic acid [Holz, G.G. Jr., Bull. W.H.O. 55:237-248 (1977)].

Further evidence for the various fatty acyl substituents was obtained from GLC-MS analysis of intact GPIs and products of specific degradations and the results are given in Figure 13. These data agree with the results of mass spectrometry.

Based on the above data, the structure shown in Fig. 7 is proposed for the P. falciparum GPIs. EXAMPLE 11

7NE- -inducing activity of P. falciparum GPIs. Previous studies have shown that P. falciparum GPIs can induce the expression of proinflarnmatory cytokines in macrophages [Schofield, L. et al., Annals Trop. Med. Parasitol. 87:617-626 (1993); Schofield, L. et al., J. Immunol 156: 1886-1896 (1996); and Tachado, S.D. et al., Proc. Natl Acad. Sci. USA 94:4022-4027 (1997)]. To determine if highly purified GPIs can induce biologic activity, cultured murine macrophages were incubated with 0.03-0.5 M purified GPIs and TΝF- in the culture medium was measured. Consistent with a previous finding [Schofield, L. et al., j. Exp. Med. 177:145-153 (1993)], the purified parasite GPIs induced TΝF- (30 to 450 pg/ml) in a dose- dependent manner (not shown).

EXAMPLE 12

Analysis for the natural anti-GPI antibody in human. To examine whether people living in malaria endemic areas express naturally elicited anti-GPI antibodies, we analyzed sera from 300 adults in Western Kenya, and 35 USA adults not exposed to malaria. In an ELISA by coating 2 ng/well HPLC purified free matured GPIs, all

Kenyan adult sera exhibited moderate to high levels of GPI-specific antibodies, predominantly IgGs, whereas all USA adults completely lacked anti-GPI antibodies (Fig. 8). The anti-GPI antibody activity of Kenyan sera was dose dependent and saturable (Fig. 8). There was no significant difference in the antibody levels of sera analyzed whether HPLC purified free and protein-linked GPIs was used for ELISA, indicating that the immunoreactivity is not due to contamination by parasite proteins.

More than 85% Kenyan adult sera also contained lower but significant levels of GPI- specific IgM antibodies (Νaik et al, unpublished results). The erythrocyte membrane debris, saponin-lysate and ghosts were nonreactive to the infected Kenyan sera (data not shown). When sera were tested by ELISA using several commercially available phospholipids, Pis from bovine liver and soybean that lack acyl substituent on inositol, PGs and CL, Kenyan adult sera showed a low level of activity; about 5-15 % of that observed with P. falciparum GPIs (Fig. 9). At all coating concentrations tested (2 to 50 ng/well), these compounds showed similar low levels of antibody reactivity. Incubation of Kenyan sera with 2.5 to 20 ng/ml of Pis, PGs, or CL prior to ELISA, using plates coated with the parasite GPIs, showed in all cases -5-15% lower antibody binding activity irrespective of the concentrations of lipid used (Fig. 8). However, prior incubation with the parasite GPIs inhibited binding up to 75% in a dose-dependent manner (Fig. 9). Together, these results suggest that Kenyan adult sera have low levels of reactivity to common acylated phosphoglycerols. This is not suφrising because of the polyclonal nature of GPI-specific antibodies, which are expected to contain antibodies to phosphoglycerol portions of the GPIs that are common to this class of molecules. TLC-immunoblotting of the total HPLC-purified GPIs confirmed the specificity of the seroreactivity; only adult Kenyan sera showed immunoreactivity to the specific GPI bands, while control sera from USA adults were nonreactive (Fig. 10). Furthermore, the Kenyan adult sera were nonreactive to bovine liver and soybean Pis that lack an acyl substituent on inositol, PGs, and CL (Fig. 10, and not shown); the apparent lack of reactivity to these lipids on TLC plates could be due to a low level of sensitivity. Furthermore, treatment of the GPIs with HNO₂, shifted the immunoreactive bands to the position of the PI moiety, suggesting that this portion of the molecule is antigenic.

The identity of the antigenic part of GPIs was further confirmed by inhibition of seroreactivity to intact GPIs using carbohydrate and lipid moieties of GPIs isolated after HNO₂ fragmentation. The carbohydrate moiety inhibited antibody binding by only -5% at a 10-fold higher concentration compared with that of the coated intact GPI. The lipid moiety, at the same coating concentration as that of intact GPIs, showed >70% antibody binding activity, indicating that the PI moiety is the antigenic structure. These results suggest that the acylated inositol is the major moiety that recognizes the naturally elicited anti-GPI IgGs.

EXAMPLE 13

Anti-GPI antibody response and acquired resistance to malaria pathogenesis.

To determine whether the susceptibility of young children in malaria endemic areas is related to the absence of GPI-specific antibodies, sera taken every month after birth up to 4 years were analyzed from a cohort of 48 children (11). The results were compared with those of sera from 100 siblings (7-8 years) and 50 non-pregnant mothers (20-25 years) (Fig. 11A). Malaria parasitemia and clinical parameters (hemoglobin and fever) were closely followed every two weeks in this community- based, prospective cohort. Whereas a case-control study design compares extremes, this non-biased design determined if an association between anti-GPI antibodies and malaria pathogenesis was detectable at a population level. We carefully controlled for P. falciparum transmission, anti-malaria drug treatment, and age. By sampling the same child over four years of life, we found that, within an individual, anti-GPI antibody responses were correlated with protection against malaria-attributable hemoglobin loss and febrile illness (see below).

We performed ELISA on sera from each child on all monthly samples and for siblings and adults at two time points spaced by 1-3 months to determine the anti-GPI antibody level and the persistence of the antibody response. For comparison and correlation analysis, results of four time points (0.5, 1.5, 2.5, and 3.5 years old), based upon the sampling scheme employed for the 7-8 year olds and the mothers, were used for children (Fig. 11 A). The data show that -50% of the children under 2 years lacked anti-GPI antibodies (negative responders, defined in legend to Fig. 11), -40% had short-lived antibody responses (lasted in the circulation for only 1-3 months, intermittent responders), and only ~10%ι had a persistent (long-lived) responses (positive responders) (Fig. 11A). By contrast, -75% of the 7-8 year-old children and all adults, exhibited a persistent anti-GPI antibody response. The level of anti-GPI IgG increased with increasing age from infancy to adulthood, while, mean hemoglobin density increased and fever (temperature) decreased with age up to 7-8 years of life (Fig. 1 IB). Thus, the presence of long-lived, high levels of anti-GPI antibodies in sera parallels the naturally acquired resistance against malaria pathogenesis.

We found that anti-GPI antibody response, age, and parasite density were independently associated with hemoglobin (p <0.0147). Age influenced the anti-GPI antibody responder category; however, it was not significant (p <0.058 1), suggesting that the association between antibody responder category (as defined in legend to Fig. 8) and hemoglobin was not just a reflection of age. For example, using a General Linear Model, it was estimated that the 6 months old children in the positive antibody responder category had 2.37 g/dl of hemoglobin more than the 6 months old in the intermittent responder category. Antibody responder category and parasite density were independently associated with temperature (p <0.0012). Six-month olds in the negative antibody responder category had an estimated temperature of 0.71 °C higher than the individuals in the intermittent antibody responder category. Fig. 12 shows the associations between anti-GPI antibody responder category and malaria- attributable pathology in children 0.5-3.5 years old. In each parasitemia category, febrile illness increased and hemoglobin level decreased in children without or with only short lived antibody; however, in children with persistent antibodies, febrile illness was lower and hemoglobin was higher. These results strongly support the hypothesis that circulating anti-GPI antibodies neutralize the toxic effects of parasite GPIs.

Conclusions

The results discussed herein establish for the first time that individuals residing in malaria-endemic areas develop a P. falciparum-specific anti-GPI antibody response, whereas people not exposed to the malaria parasite do not have these antibodies; in addition, anti-GPI antibody responses are correlated with protection against malaria-related febrile illness and hemoglobin loss. This agrees with the previous reports describing P. falciparum GPIs as pathogenicity factors based on their ability to induce a spectrum of proinflammatory cytokine responses and cause malaria symptoms. Another important finding is elucidation of complete structures of the parasite

GPIs by direct biochemical analysis and mass spectrometry. Particularly, our results identified two novel structural features of P. falciparum GPIs: (a) the presence of palmitate and myristate on C-2 of inositol, and (b) the presence of oleic and/or cis- vaccenic acid at sn-2. The purification of P. falciparum GPIs to homogeneity was crucial for structural determination as well as to obtain unambiguous evidence for biologic activity and establish the presence and specificity of naturally elicited antibodies in sera of people living in malaria endemic areas. The key steps employed for isolation and purification of GPIs were: (a) growing of mycoplasma-free cultures to high levels of parasitemia and enrichment of infected erythrocytes; (b) metabolic labeling of GPIs and the use of human sera containing anti-GPI antibodies to follow purification steps; (c) use of sterile water and buffers, and high quality organic solvents to exclude external contamination; (d) siliconizing the glasswares to avoid loss due to surface adsoφtion. The mass spectrometry results presented here show that the purified GPIs are homogeneous.

We determined the structures of P. falciparum GPIs by subjecting the [ H]GlcN- and [ H]fatty acid-labeled GPIs to various standard degradative procedures including their susceptibility to nitrous acid, HF, alkali, jack bean -mannosidase, GPI-specific phospholipase D, fatty acid compositional analysis, and finally by direct mass spectrometry. The results enabled us to propose the structures shown in Fig. 6 for the parasite GPIs. The structure of the core glycan is the same as previously determined [Gowda, D.C. et al., J. Biol. Chem. 272:6428-6439 (1997); Gerold, P. et al., J. Biol Chem. 269:2597-2606 (1994)]. However, the structures are different from those previously reported, based on radiolabeling studies, with regard to acyl substituents that identified palmitate at both sn-l and sn-2, and predominantly myristate on inositol [Gerold, P. et al., Mol. Biochem. Parasitol. 75:131-143 (1996)]. As shown in Fig. 8, palmitate is the major acyl substituent with minor proportions of myristate on inositol of the parasite GPIs; GPIs with acylated inositol residues from other sources reported to date contain only palmitate on the inositol residue [Ferguson, M.A.J. et al., Biochim. Biophys. Ada. 1455:327-340 (1999)]. The parasite GPIs contain exclusively an unsaturated acyl substituent at sn-2 (major C18:l and minor C18:2), and a range of variable size saturated acyl residues at sn-l. With respect to the nature of acyl residue at sn-2, the parasite GPIs resemble the GPI of Trypanosoma cruzi trypomastigote mucin that has potent cytokine-inducing property [Almeida, I.C. et al., EMBO J. 19:1476-1485 (2000)]. Another unusual feature of the parasite GPIs is the likely presence of cz^'s-vaccenic acid at sn-2 as suggested by the incoφoration of radiolabeled palmitoleate at sn-2.

The parasite GPIs significantly differ from those of man with respect to both the acyl substituents, the carbohydrate moiety and/or the type of substitution on carbohydrate [Ferguson, M.A.J. et al., Biochim. Biophys. Ada. 1455:327-340 (1999)]. The GPI moieties of human erythrocyte (which do not contain detectable levels of free GPIs) proteins, CD59 and acetylcholine esterase, contain exclusively a C18 alkyl substituent at sn-l, C22:4 at sn-2, and palmitate on inositol; the carbohydrate moieties contain 1 or 2 extra phosphoethanolamine as well as G a IN Ac residues [Ferguson, M.A.J. et al., Biochim. Biophys. Ada. 1455:327-340 (1999)]. The GPI of human spleen CD52 contains a diacylglycerol moiety and lacks GalNAc; however this differs from the parasite GPIs with respect to the type of fatty acid at sn-2 (C22:4, C22:5, and C22:6), and contains phosphoethanolamine on the first Man residue [Ferguson, M.A.J. et al., Biochim. Biophys. Ada. 1455:327-340 (1999)]. These structural differences may contribute to the observed naturally elicited immunologic responses against the parasite GPIs in man.

Although Schofield et al. have shown that P. falciparum GPIs can transduce signals to elicit inflammatory cytokine responses [Schofield, L. et al., Annals Trop. Med. Parasitol 87:617-626 (1993); Schofield, L. et al., J. Immunol 156: 1886-1896 (1996); and Tachado, S.D. et al., Proc. Natl Acad. Sci. USA 94:4022-4027 (1997)], there have been concerns as to whether the observed activity was due to contamination (parasite, erythrocytes, and/or mycoplasma origin) [Taverne, j. et al., Parasitol. Today. 13:276-277 (1997)]. These concerns were based on the observations by some investigators that aqueous buffer extracts of parasite cultures, that presumed to have extracted the parasite GPIs upon boiling, could not elicit TNF- . Thus, they have argued that thecytokine-inducing property of P. falciparum was due to unknown components. However, it should be noted that GPIs can only be extracted with organic solvents. Because of this existing controversy, we tested TNF- induction by the highly purified parasite GPIs. The results presented in this paper clearly show that the purified P. falciparum GPIs can induce TNF- i n macrophages.

This activity is consistent with the recent finding by Almeida et al. that a highly purified GPI moiety of T. cruzi trypomastigotes mucin induces TNF- [Almeida, I.C. et al, EMBO J. 19:1476-1485 (2000)] and confirms the previous finding by Schofield et al. [Schofield, L. et al., J. Exp. Med. 177:145-153 (1993)]. Furthermore, studies by Almeida et al. also show that C18:l and/or C18:2 acyl substituent at sn-2 in the T. cruzi GPIs is critical for activity (30). Since the P. facliparum GPI also contains C18:l (major) and C18:2 (minor) at sn-2, it is possible that these acyl substituents contribute to the toxic property of the parasite GPIs.

In malaria endemic areas, younger children have the highest risk of developing severe malaria, whereas older children and adults rarely develop severe disease despite repeated exposures and significant parasitemia [Hommel, M., Annals Trop. Med. Parasitol. 87:627-635 (1993)]. People in non-malarious regions completely lack this resistance, suggesting that the protection is due to a parasite-specific response acquired through repeated infections. In the Western Kenyan population studied here, we found that adults have P. falciparum parasitemia >14% of the time, whereas children <4 years old have parasitemia >60% of the time. The density of parasitemia is lower in adults and, importantly, the level of parsitemia that can be tolerated without causing febrile illness or anemia is higher in adults (OraLee et al. unpublished). Since P. falciparum GPIs are pathogenicity factors [Schofield, L. et al., J. Exp. Med. 177:145-153 (1993); Schofield, L. et al., Annals Trop. Med. Parasitol. 87:617-626 (1993); Schofield, L. et al., J. Immunol 156: 1886-1896 (1996); and Tachado, S.D. et al., Proc. Natl Acad. Sci. USA 94:4022-4027 (1997)], the resistance of adults to malaria illness may be related to a GPI-specific protective immunity.

All the Kenyan adult sera analyzed contained high levels of GPI-specific IgGs, whereas all 50 USA adult sera completely lacked such antibodies. The antibody response was highly specific to GPIs and their intermediate; other phospholipids of the parasite showed only low levels of immunoreactivity. Several phospholipids, including Pis, PGs, and cardiolipin, showed only 5-15% of the immunoreactivity exhibited by GPIs, which appears to be due to the polyclonal nature of the antibodies reacting with the common epitopes. Previously, several studies have reported the presence of significant levels of antibodies that bind phospholipids either directly or via binding of serum 2-glycoprotein I [Jakobsen, P.H. et al. , Clin. Exp. Immunol. 105:69-73 (1996)]. In those studies, lipids were coated with several g/w ell for ELISA. It is known that proteins and antibodies can nonspecifically bind to lipids when coated at high density. In contrast, our study used 0.5 to 2 ng/well for ELISA and 100 ng for TLC-immunoblots. Moreover, in previous studies, plates coated with lipid antigens were incubated with sera diluted with buffers without detergent. Under such conditions, we found high levels of nonspecific activity. Thus, our study clearly demonstrates that the identified IgGs are specific to GPIs.

Our results also establish that the PI portion of the GPIs contributes significantly to the immunogenicity. The removal of the sn-2 fatty acid from the GPIs did not affect antibody reactivity (Vijayakumar et al, unpublished results). Since treatment with HF abolished immunoreactivity, it appears that the acylated-inositol phosphate is the immunogenic portion of the molecule. This agrees with the previous finding that antibodies raised against Pis can inhibit the induction of TNF- b y P. falciparum extracts [Bate, C.A.W. et al., Infect. Immun. 62:5261-5266 (1994)]. These results are important in that if an sn-2 acyl substituent is indeed required for cytokine- inducing activity, then it may be possible to synthesize non-toxic molecules for therapeutic puφoses.

The TNF- -inducing activity of GPIs and the general resistance of adults in endemic areas to malaria pathogenesis and its correlation to the presence of serum anti-GPI antibody response suggest that the acquired immunity is related significantly to the anti-GPI antibodies. This prediction agrees with lack of such an antibody response in the majority of the children <4 years, risk of children developing severe malaria, and the correlation between the gradual acquisition of the antibody response in an age-dependent manner (>80% of the 7 to 8-years old having high levels of serum antibody) and protection against malaria with age. Thus, a direct correlation between anti-GPI antibodies and malaria-related pathology could be observed in young children.

High levels of antibodies against several other antigens such as MSP-1, EBA- 175, and circumsporozoite protein are also present in adults, and therefore it may be argued that the anti-GPI antibody response is not independent from those antibodies. However, it is important to consider that the anti-GPI antibody response is related to anti-disease immunity, whereas antibodies against parasite proteins studied to-date are involved in anti-parasite immunity (control parasite burden). Furthermore, whereas -80% of children <2 years of age either do not have or contain very low levels of short lived anti-GPI antibodies, children <2 years of age can have high levels of antibodies against MSP-1 and other proteins [Branch, O.H. et al., J. Infect. Dis. 181:1746-1752 (2000); Branch, O.H. et al., Am. J. Trop. Med. Hyg. 58:211-219 (1998); and OraLee et al. (unpublished results)]. Although there was a correlation between the levels of MSP-1 and EBA-175 antibodies and protection against parasite density, these antibody responses were not related to the anti-GPI antibody response, protection against febrile illness, and hemoglobin loss at any given parasite density.

Almost all malaria vaccine development efforts currently being pursued use parasite proteins in a multi-component formulation, aimed at providing immunity against infection (anti-parasitic) [Shi, Y.P. et al., Proc. Natl Acad. Sci. USA 96:1615- 1620 (1999)]. By contrast, blocking the toxic effects of the parasite GPIs, a GPI-based vaccine may significantly reduce malaria pathogenesis. The identification of the PI moiety as the functional part of the molecules will aid in the development of effective anti-malaria disease measures.

All references discussed above are hereby incoφorated by reference in their entirety.

Claims

CLAIMSWe claim:

1. A method for treating or preventing malaria in a subject comprising the step of administering a therapeutically or prophylactically effective amount of an agent which inhibits Plasmodium glycosylphosphatidylinositol (GPI) biosysnthesis or which inhibits Plasmodium GPI activity in the host.

2. The method of treating malaria of claim 1, wherein the agent is administered in combination with at least one other antimalarial compound.

3. The method of treating malaria of claim 2, wherein the antimalarial compound is doxycycline, a dihydrofolate inhibitor, sulfonamide, sulfones, tetracyclines, chloroquine, quinine, quinidine, amodiaquine, halofantrine or mefloquine.

4. The method of claim 3, wherein the dihydrofolate inhibitor is pyrimethamine or proguanil.

5. The method of claim 1, wherein the Plasmodium GPI is selected from the group consisting of a Plasmodium falciparum GPI, a Plasmodium malariae GPI, a Plasmodium vivax GPI and a Plasmodium ovale GPI.

6. The method of claim 5, wherein the GPI is a P. falciparum GPI and is a GPI found on one of the following proteins MSP- 1, MSP-2, MSP-4, p71, 55 kDa merozoite rhoptry antigen, 102 kD transferrin receptor, and a 76 kD seine protease.

The method of claim 5, wherein the GPI is:

and wherein n is 12, 14, 16, 18 or 20, Rj is C₁₅H₃₁ or C₁₃H₂ and R₂ is CπH₃₃ or C₁7H₃₁.

8. The method of claim 7, wherein n is 16.

9. The method of claim 5, wherein the GPI is a P. falciparum GPI and is:

wherein Ri is myristic acid, Cι₅H₃j, Cι₃H₂₇ or palmitic acid; R₃ is oleic acid, cis- vaccenic acid, linoleic acid, C₁ H₃ or C₁₇H₃₁; R₂ is CH₃(CH₂)n-; and n = 12, 14, 16, 18 or 20.

10. The method of claim 9, wherein n is 16.

11. The method of treating malaria of claim 1 , wherein the agent which inhibits GPI biosynthesis is: GlcN, mannosamine, benzyl-α-2-amino-2-deoxy- glucosaminide, phenyl-α-2..amino-2.deoxyglucosaminide or aryl-2-amino-2-deoxy- glycosaminide.

12. The method of treating malaria of claim 1, wherein the agent which inhibits malaria binds to the Plasmodium GPI and wherein said binding blocks GPI- mediated pathology.

13. The method of claim 12, wherein the agent which binds to the Plasmodium GPI is an antibody which inhibits GPI activity.

14. The method of claim 13, wherein the antibody is a chimeric, bispecific, human or humanized antibody.

15. The method of preventing malaria of claim 1, wherein the agent which prevents malaria comprises GPI or a fragment thereof.

16. The method of claim 15, wherein the GPI fragment is selected from the group consisting of

(I)

Ino-O-CO

I I

OP-O- R_j

O

CH₂ -CH-CH₂

1 f

0 0

|

1 i CO c=o

or (II) GlcNαl- Iπo-0-C=0

1 I

0=P-CV R.

1 I 0

1

CH₂ -CH-CH₂

1 I

0 o

I 1 oo c=o

1 I 1

1

R2 R₃

wherein Ri is myristic acid, C₁₅H₃₁, CπH₂₇ or palmitic acid; R3 is oleic acid, cis- vaccenic acid, linoleic acid, C₁₇H₃₃ or Cι₇H_3!; R₂ is CH₃(CH₂)n-; and n = 12, 14, 16, 18 or 20.

17. The method of claim 16, wherein n is 16.

18. A pharmaceutical composition for treating or preventing malaria in a subject comprising a Plasmodium GPI or an immunogenic fragment thereof and a pharmaceutically acceptable carrier.

19. The pharmaceutical composition of claim 18, wherein the immunogenic fragment of GPI is:

(I) Ino-0-C=0

I I

I o i

CH CH-CH₂

I - I - ..

0 o

1 - I-

00 co

or

4GlcNαl- Ino-0-C=0

(II) 1 I

0=P-0 R,

1

0

J

CH₂ -CH-CH₂

1 I o 0

1 1

OO CO t 1

R* R₃

wherein Ri is myristic acid, Cι₅H₃₁, Cι₃H₂ or palmitic acid; R₃ is oleic acid, cis- vaccenic acid, linoleic acid, C₁₇H₃₃ or Cι₇H₃₁; R₂ is CH₃(CH₂)n-; and n = 12, 14, 16, 18 or 20.

20. The pharmaceutical composition of claim 18, wherein GPI is:

wherein Ri is myristic acid, Cι₅H ₁, C₁₃H₂₇ or palmitic acid; R₃ is oleic acid, linoleic acid, C H₃₃ orC₁₇H₃₁; R₂ is CH₃(CH₂)n-; and n = 12, 14, 16, 18 or 20.

21. The pharmaceutical composition of claim 20, wherein n is 16.

22. The composition of claim 18, wherein said Plasmodium GPI is selected from the group consisting of P. falciparum, P. vivax, P. ovale and P. malariae.

23. A method of diagnosing whether a person has antibodies to malaria comprising:

(A) obtaining a sample from a subject; (B) exposing said sample to Plasmodium GPI or an immunogenic fragment thereof; and

(C) assaying for whether said Plasmodium GPI or an immunogenic fragment thereof binds an antibody.

24. A kit for determining whether a person has antibodies to malaria comprising a Plasmodium GPI or immunogenic fragment thereof; wherein the GPI or immunogenic fragment is attached to a detectable label.

25. A method of identifyiing an agent that binds to a malarial GPI and thereby inhibits GPI-mediated pathology comprising the steps of:

(A) adhering a Plasmodium GPI to a substrate;

(B) exposing said substrate to a candidate compound;

(C) determining whether said candidate compound binds to GPI; and

(D) assaying said candidate compound for GPI-inhibiting activity.

26. A method of diagnosing whether a person has antibodies to malaria comprising:

(A) obtaining a sample from a subject;

(B) exposing said sample to Plasmodium GPI or an immunogenic fragment

(C) assaying for whether said Plasmodium GPJ or an immunogenic fragment thereof binds an antibody.

27. A kit for determining whether a person has antibodies to malaria comprising a Plasmodium GPI or immunogenic fragment thereof; wherein the GPI or immunogenic fragment is attached to a detectable label.

28. A vaccine comprising at least one of the following:

Iπo-O-C=O

I I

C P-O- R, f (II) O

I

CH₂-CH-CH₂ i I

0 O

1 I

C=O CO

R₂ R₃ _(m) GlcNαl- Ino-O-CO

I I 0=P-0- R,

I- -- o

wherein R_! is myristic acid, C_l5H₃i, CπH₂₇ or palmitic acid; R₃ is oleic acid, cis- vaccenic acid, linoleic acid, C_l7H₃₃ or C₁7H₃₁; R₂ is CH₃(CH₂)n-; and n = 12, 14, 16, 18 or 20.