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Infection and Immunity, February 2006, p. 1412-1415, Vol. 74, No. 2
0019-9567/06/$08.00+0     doi:10.1128/IAI.74.2.1412-1415.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Naturally Elicited Antibodies to Glycosylphosphatidylinositols (GPIs) of Plasmodium falciparum Require Intact GPI Structures for Binding and Are Directed Primarily against the Conserved Glycan Moiety

Ramachandra S. Naik,1,{dagger} Gowdahalli Krishnegowda,1,2 Christian F. Ockenhouse,3 and D. Channe Gowda1,2*

Department of Biochemistry and Molecular Biology, Georgetown University Medical Center, Washington, D.C. 20007,1 Department of Biochemistry and Molecular Biology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033,2 Department of Immunology, Walter Reed Army Institute of Research, Silver Spring, Maryland 209103

Received 29 September 2005/ Returned for modification 1 November 2005/ Accepted 10 November 2005


   ABSTRACT
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Immunization with a synthetic glycan corresponding to Plasmodium falciparum glycosylphosphatidylinositols (GPIs) has been proposed as a vaccination strategy against malaria. We investigated the structural requirements for binding of naturally elicited anti-GPI antibodies to parasite GPIs. The data show that anti-GPI antibody binding requires intact GPI structures and that the antibodies are directed predominantly against GPIs with a conserved glycan structure with three mannoses and marginally against the terminal fourth mannose. The results provide valuable insight for exploiting GPIs for the development of malaria vaccines.


   TEXT
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Plasmodium falciparum glycosylphosphatidylinositols (GPIs) have been shown to induce the production of proinflammatory cytokines in macrophages and to cause symptoms reminiscent of acute malaria in mice (1, 7, 9, 12, 15). Thus, GPIs have been identified as the prominent toxin that contributes to malaria pathogenesis. A monoclonal antibody to P. falciparum GPIs has been reported to neutralize the tumor necrosis factor alpha (TNF-{alpha})-inducing activity of GPIs, suggesting that naturally elicited anti-GPI antibodies can provide protection against malaria pathogenesis (13). During the past few years, we and others have shown that people in areas where malaria is endemic produce anti-GPI antibodies in an age-dependent manner (2, 3, 5, 8, 10, 11). Adults and adolescents with protective immunity to malaria have persistently high levels of antibodies, whereas children aged <3 years either lack or have low levels of antibody. Anti-GPI antibodies were found to be primarily of the short-lived immunoglobulin G3 (IgG3) subclass, with lower levels of IgG1 (4). In a population in Western Kenya, where malaria is endemic, the anti-GPI antibody responses were found to be associated with protection against malarial anemia and fever (10). A more recent study involving people in Senegal suggested that anti-GPI antibodies are involved in protection against cerebral malaria (11). However, in several other studies, the role of anti-GPI antibodies in protecting the host against malaria pathology was not clearly evident (3). Further-defined, case-controlled studies are needed to define the role of anti-GPI antibodies in protection against malaria pathogenesis. Nevertheless, it was recently reported that mice immunized with a synthetic glycan corresponding to the P. falciparum GPIs were substantially protected against Plasmodium berghei-induced cerebral malaria (14). These results also demonstrate that vaccination with P. falciparum GPIs or their components can be a strategy for preventing severe malaria (14). Therefore, understanding the relationship between GPI structures and anti-GPI antibody-binding activities is valuable in the rational design of GPI-based vaccine candidates.

This study was undertaken to determine the epitope specificities of naturally occurring anti-GPI antibodies in people living in areas where malaria is endemic, using GPIs purified from P. falciparum and chemically defined structural fragments of GPIs (Fig. 1), which were prepared and characterized as described previously (10, 16). Briefly, P. falciparum parasites released by 0.05% saponin were purified by density centrifugation on cushions of 5% bovine serum albumin and lyophilized. The GPIs were extracted with chloroform-methanol-water (10:10:3 [vol/vol/vol]) and purified by residue partitioning between water and water-saturated 1-butanol followed by reversed-phase high-performance liquid chromatography (HPLC) using a C4 Supelcosil column (10). Man3-GPIs (GPIs lacking the terminal fourth mannose residue) and sn-2 lyso-GPIs (GPIs lacking a fatty acid substituent at the sn-2 position) were prepared by treatment of the purified P. falciparum GPIs with jack bean {alpha}-mannosidase and bee venom phospholipase A2, respectively, and were purified by HPLC (10, 16). The glycan lacking phosphatidylinositol (PI) moiety was prepared by treatment of the purified GPIs with HNO2 (10, 16). The glycan containing acylated inositol and diacylglycerol moiety was obtained by treatment of GPIs with aqueous HF (10, 16). Deacylated GPI was prepared by incubating the GPIs with 1 M ammonium hydroxide in 50% aqueous methanol (1:1 [vol/vol]) at 37°C for 12 h. The purified GPIs, modified GPIs, and GPI glycan fragments were quantitated by determining their mannose and glucosamine contents by high-pH anion-exchange HPLC of samples hydrolyzed with 2.5 M trifluoroacetic acid at 100°C for 4 h (for mannose) or 3 M HCl at 100°C for 3 h (for glucosamine) (6).


Figure 1
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  		FIG. 1. Structures of P. falciparum GPIs, modified GPIs, and GPI fragments. The structure of the intact GPI (Man4-GPI) purified from the parasite and the enzymatic and chemical cleavage sites are indicated in the top panel. R1, R2, and R3 are fatty acid substituents. R1 is predominantly C18:0 and minor amounts of C16:0, C14:0, C20:0, and C22:0; R2 is C18:1 (major) and C18:2 (minor); R3 is C16:0 (major) and C14:0 (minor). The arrowhead shows the positions of cleavage by NH4OH.

 
Previous studies have shown that adults in Western Kenya and other malaria holoendemic areas have persistently high levels of anti-GPI antibodies (2, 5, 8, 10, 11). In this study, we evaluated the epitope specificities of the naturally elicited circulatory antibodies in the sera of 10 healthy adults from Western Kenya. Sera from five healthy U.S. adults were used as controls. In an enzyme-linked immunosorbent binding assay (10), we assessed in parallel the serum antibody binding abilities of the GPIs (Man4-GPIs) purified from P. falciparum and GPIs lacking either the terminal fourth mannose residue (Man3-GPIs) or the fatty acid substituent at the sn-2 position (sn-2 lyso-GPIs), which were used to coat wells at concentrations ranging from 0.01 to 4 ng GPIs/well (Fig. 2). Consistent with the results of a previous study (10), the anti-GPI antibodies in sera of all individuals from the area of malaria endemicity could bind to the purified intact GPIs as well as the modified GPIs, Man3-GPIs and sn-2 lyso-GPIs, in a coating-concentration-dependent manner, with saturated levels of binding at 2 ng/well GPIs (Fig. 2A). In contrast, the sera from the individuals not exposed to malaria parasites (the healthy U.S. controls) showed only background levels of binding to GPIs at all coating concentrations. These results indicate that the bound serum antibodies in malaria parasite-exposed individuals are directed against GPIs. In sera from all the malaria parasite-exposed individuals, the anti-GPI antibody binding capacities of Man3-GPIs and sn-2 lyso-GPIs were consistently 4 to 22% lower than those of the intact GPIs (Man4-GPIs) (Fig. 2B). The observed difference in antibody binding capacity between Man4-GPIs and Man3-GPIs and that between Man4-GPIs and sn-2 lyso-GPIs were statistically significant (P < 0.05; Student's t test). However, in each case, only 4 of 10 sera from malaria-exposed individuals showed binding (Fig. 2B). These results demonstrate that the terminal fourth mannose and sn-2 fatty acid residue contribute only marginally to the antigenicity of the parasite GPIs.


Figure 2
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  		FIG. 2. Analysis of anti-GPI antibodies in sera from adults in areas where malaria is endemic. Purified P. falciparum GPIs, Man3-GPIs, and sn-2 lyso-GPIs were used to coat 96-well microtiter plates at 0.01 ng to 4 ng per well. The wells were blocked with casein and then incubated with sera diluted 1:200. The bound antibodies were measured using horseradish peroxidase-conjugated goat anti-human IgG (heavy and light chains), with 2,2'-azinobis(3-ethylbenzthiazolinesulfonic acid) (ABTS) as the color substrate. Assays were carried out two times each in triplicate, and mean values were plotted. (A) GPI coating-concentration-dependent binding curves for representative sera from a Western Kenyan (solid lines) and an American control individual (dashed lines). (B) Anti-GPI antibody binding to Man4-GPIs, Man3-GPIs, and sn-2 lyso-GPIs (each used at 4 ng/well) in sera from malaria-exposed individuals. Significant differences in anti-GPI antibody binding to Man3-GPIs or sn-2 lyso-GPIs compared to binding to Man4-GPIs are indicated by asterisks (P < 0.05).

 
To determine whether anti-GPI antibodies are directed against the glycan or PI portion of GPIs, we analyzed the sera of the malaria-exposed individuals for inhibition of antibody binding by the glycan and lipid moieties in an enzyme-linked immunosorbent assay (Fig. 3). In parallel, sera were also evaluated for inhibition of antibody binding to parasite GPIs by unmodified GPIs and modified GPIs. In all sera, the intact GPIs could inhibit the binding of antibodies to GPI-coated plates in a dose-dependent manner, with 60 to 80% inhibition by 2.5 to 20 ng/ml GPIs in solution (Fig. 3). The Man3-GPIs and sn-2 lyso-GPIs could also efficiently inhibit antibody binding to intact GPIs; their inhibitory capacity was about 10 to 20% lower than that of intact GPIs. These results demonstrate that the anti-GPI antibodies are directed predominantly against the conserved glycan structure, comprising three mannoses, of the GPIs. In contrast to these results, the glycan moieties obtained by removal of the PI portion or the diacylglycerol phosphate residue could not inhibit antibody binding to GPIs (Fig. 3). The deacylated GPI moiety obtained by the treatment of GPIs with NH4OH was also unable to inhibit anti-GPI antibody binding to GPIs (Fig. 3). The lipid moiety (PI) of the parasite GPIs showed only a marginal inhibition of antibody binding to intact GPIs, with about 5 to 18% inhibition at 2.5 to 20 ng/ml. A mixture of the PI and glycan moieties was also inefficient in inhibiting antibody binding to GPIs, with about 20% inhibition at 20 ng/ml. Taken together, these results indicate that the binding of anti-GPI antibodies by GPIs requires the intact GPI structure.


Figure 3
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  		FIG. 3. Inhibition of serum anti-GPI antibody binding to P. falciparum GPIs by modified GPIs and by the glycan and lipid moieties of GPIs. Ninety-six-well microtiter plates were coated with the purified intact GPIs at 2 ng/well, blocked with casein, and incubated with sera diluted 1:200 and pretreated for 30 min separately with intact GPIs, modified GPIs, or glycan or lipid components. The bound antibodies were measured using horseradish peroxidase-conjugated goat anti-human IgG (heavy and light chains) and the ABTS color substrate. Assays were performed two times each in duplicate, and average values were plotted. The results shown are for a representative Western Kenyan serum.

 
The observed dual requirement of the glycan and lipid moieties of intact GPIs for anti-GPI antibody binding resembles our previous finding that the inflammatory cytokine-inducing activity of GPIs requires the simultaneous recognition of the glycan and lipid moieties of GPI molecules (16). The glycan and lipid moieties of the parasite GPIs either alone or in a mixture were unable to produce TNF-{alpha} in macrophages (16). These results, together with those of the present study, demonstrate that the glycan and PI moieties of intact GPIs are involved in the induction of both innate and adaptive immune responses.

It was recently reported that mice immunized with a synthetic glycan, EtN-P-(Man{alpha}1-2)6Man{alpha}1-2Man{alpha}1-6Man{alpha}1-4GlcN{alpha}1-6-myo-inositol-1,2-cyclic phosphate, which represents the glycan portion of P. falciparum GPIs, were protected substantially from severe malaria and fatality when challenged with P. berghei ANKA (14). Antibodies produced against the synthetic glycan could neutralize the proinflammatory activity of P. falciparum GPIs. These results, when considered with our observation that the monovalent glycan moieties of parasite GPIs were unable to inhibit serum antibody binding to GPIs, suggest that the anti-GPI antibodies produced by mice immunized with the synthetic glycan-keyhole limpet hemocyanin conjugate are directed specifically against multivalent glycan clusters in the conjugate. Therefore, it is likely that the antibodies in mice immunized with the synthetic glycan-keyhole limpet hemocyanin conjugate bind clusters of exposed hydrophilic glycan moieties of GPIs presented by the parasite, neutralizing the GPIs' toxic activity. Furthermore, it appears that the clusters of hydrophilic glycan moieties of GPIs are important for immune responses and that the lipid moieties of the GPIs are critical in the context of presenting the glycans as multivalent clusters.

In conclusion, in this study we define for the first time the structural requirements for the binding of naturally elicited anti-GPI antibodies to GPIs purified from P. falciparum. The data demonstrate the dual requirement of the glycan and lipid moieties of intact GPIs for antibody binding. Our data also show that anti-GPI antibody responses are directed mainly against the conserved GPI structure with three mannose residues and a lipid moiety. These results will be valuable in designing GPI-based vaccine candidates.


   ACKNOWLEDGMENTS
 
We thank Brian de Souza, Royal Free and University College London Medical School, London, United Kingdom, for helpful comments on the manuscript.

This work was supported by grant AI41139 from NIAID, NIH.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, Pennsylvania State University College of Medicine, 500 University Dr., Hershey, PA 17033. Phone: (717) 531-0992. Fax: (717) 531-7072. E-mail: gowda{at}psu.edu. Back

Editor: W. A. Petri, Jr.

{dagger} Present address: Department of Molecular Pharmacology, Division of Biochemistry, Walter Reed Army Institute of Research, Silver Spring, MD 20910. Back


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Infection and Immunity, February 2006, p. 1412-1415, Vol. 74, No. 2
0019-9567/06/$08.00+0     doi:10.1128/IAI.74.2.1412-1415.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.




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