INTRODUCTION

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The need for an effective malaria vaccine or additional therapies against the human malaria agent Plasmodium falciparum is increasing as existing control measures are jeopardized by the spread of drug resistance. An attractive target for vaccine therapy is the parasite's erythrocytic stage, which is responsible for clinical disease. In the erythrocytic stage of the life cycle, merozoites released from rupturing schizonts must invade erythrocytes within minutes to continue development. A P. falciparum ligand involved in this process is the 175-kDa erythrocyte binding protein, EBA-175 (4, 11, 13). EBA-175 attaches to erythrocytes by a sialic acid-dependent binding to its receptor, glycophorin A (14). This binding involves recognition of both the sialic acids and the peptide backbone of glycophorin A (14). The erythrocyte binding region of EBA-175 is a 616-amino-acid region, designated region II, that lies in the amino-terminal third of the molecule. Region II has a cysteine-rich motif that is also present in the Duffy-binding proteins of Plasmodium vivax and Plasmodium knowlesi (1, 2). Region II appeared to be conserved across 16 different P. falciparum strains studied (with an amino acid identity greater than 98.2%) (9).

It has been observed that the ability of native EBA-175 to bind to susceptible erythrocytes, normal or neuraminidase-treated human erythrocytes devoid of sialic acids, generally correlated closely with the ability of these erythrocytes to be invaded by P. falciparum (4, 11). However, for some P. falciparum strains, an alternative invasive pathway exists through which these strains are able to invade neuraminidase-treated erythrocytes, although with decreased efficiencies. For example, the 7G8 strain of P. falciparum invaded neuraminidase-treated erythrocytes at >50% of the level for normal erythrocytes, while the Camp strain was inhibited to >95% of the control level. Furthermore, invasion of M^kM^k erythrocytes that lack both glycophorins A and B by 7G8 strain parasites was unaffected by treatment with neuraminidase but was reduced by treatment with trypsin (>80%) (7). Given the presence of P. falciparum strains that can invade using differing ligand requirements or through pathways that are independent of an interaction with sialic acids on erythrocytes in vitro, a potential for alternative invasive pathways exists in field isolates of P. falciparum. This possibility has confounded the belief that antibodies against region II can interfere with parasite invasion in the field adequately for the targeting of EBA-175 as a vaccine candidate to be effective. Hence, the question arises: what would be the impact of a vaccine against EBA-175 region II on strains that may invade independently of sialic acids (i.e., an alternative invasive pathway)? To address this question, we selected two parasite strains, one (3D7) that invades neuraminidase-treated erythrocytes, which are devoid of sialic acids (5), and another (FVO) that is dependent on sialic acids for erythrocyte invasion, and then studied the effect of anti-region II antibodies on 3D7 and FVO parasite development in vitro. We report here that both P. falciparum strains, which have the capability to invade erythrocytes by distinct pathways, were similarly blocked by antibodies against EBA-175 region II.

	MATERIALS AND METHODS

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Parasites. Cloned 3D7 (human challenge strain) and FVO (Vietnam isolate adapted to Aotus monkeys) strains of P. falciparum were cultured and synchronized by temperature cycling through 37, 40, and 17°C (8). Schizont-infected erythrocytes were Percoll purified for analysis of merozoite invasion of enzymatically treated erythrocytes.

Erythrocytes and enzyme pretreatments. Human blood was collected in a 10% (final concentration) citrate-phosphate-dextrose solution for enzymatic treatment of erythrocytes or obtained from the Interstate Blood Bank (Memphis, Tenn.) for growth inhibition assays. The blood was stored at 4°C. Erythrocytes were washed and treated with 0.2 U of Vibrio cholerae neuraminidase (Gibco BRL, Gaithersburg, Md.) per 10⁹ erythrocytes as previously described (5) or were treated with 1 mg of trypsin (Sigma, St. Louis, Mo.) per ml essentially as previously described (4). The enzymatically treated erythrocytes were washed thrice in 100× (vol/vol) packed erythrocytes-RPMI 1640 prior to their use in parasite invasion studies.

Generation of EBA-175 region II antibodies and antibody purification. New Zealand White rabbits were immunized thrice at 4-week intervals with an EBA-175 region II DNA vaccine (FVO strain sequence) (B. K. Sim, D. L. Narum, H. Liang, et al., unpublished data) and then boosted with a homologous purified recombinant baculovirus EBA-175 region II protein (D. L. Narum, H. Liang, S. R. Fuhrmann, T. Luu, and B. K. L. Sim, unpublished data) in Freund's adjuvant. Control rabbits received plasmid without any insert and were boosted with Freund's adjuvant in phosphate-buffered saline (PBS). Polyclonal antibodies were purified by protein G column chromatography (Pharmacia, Piscataway, N.J.) using the ImmunoPure buffer system (Pierce, Rockford, Ill.).

Growth inhibition studies. Normal or enzymatically treated erythrocytes and mature Percoll-purified schizont-infected erythrocytes were added together to make a final 0.5% parasitemia in a 1 to 2% hematocrit. The parasite suspensions were plated in triplicate in 96-well flat-bottom tissue culture plates and maintained as described previously (15). Approximately 30 h postinvasion, the culture plate was centrifuged at 180 × g for 2 min and culture supernatants were removed. The erythrocytes were resuspended in 200 µl of a 1/1,000 dilution of Hydroethidine fluorescent vital stain (stock 10 mg/ml in dimethyl sulfoxide) (Polysciences, Inc., Warrington, Pa.) in PBS and incubated for 20 min at 37°C, similar to the method described previously (12). The cells were washed two times in PBS as described above and then resuspended in PBS-0.2% glutaraldehyde and stored at 4°C. Prior to analysis by flow cytometry (FACSORT; Becton-Dickinson, San Jose, Calif.) with Lysis II software, the samples were microcentrifuged, the supernatant was discarded, and the cells were resuspended in PBS. The results obtained by flow cytometry were confirmed by scoring coded Giemsa-stained thin blood films (data not shown).

	RESULTS AND DISCUSSION

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EBA-175 region II has been defined as the ligand that binds to sialic acids on its receptor, glycophorin A, on erythrocytes for invasion. Vaccine strategies involving EBA-175 region II have been confounded by the fact that in in vitro studies, P. falciparum strains are able to use pathways that are do not involve sialic acids for erythrocytic invasion (5, 7, 10). For example, Hadley et al. (7) reported different levels of invasion of neuraminidase-treated erythrocytes, which have their sialic acid residues cleaved, by two P. falciparum strains, Camp and 7G8, which were inhibited by >95% and <50% compared to the normal controls. The different levels of inhibition observed for P. falciparum FVO parasite invasion (>99.9%) and 3D7 parasite invasion (about 30%) compared to controls (Table 1) is thus another example of the differences in sialic acid requirements for invasion by different strains of P. falciparum. Thus, FVO invasion of erythrocytes involves sialic acids while 3D7 may use an alternative pathway that does not include sialic acids. Another enzyme that has been used for the analysis of erythrocytic ligand-receptor interactions is trypsin (3, 5). Invasion of trypsin-treated erythrocytes by the 7G8 strain was inhibited by 96%, whereas another strain, FCR3, which required sialic acid for invasion, was only inhibited by 64% compared to the normal controls (6). In this study, we used FVO and 3D7, whose invasion of trypsin-treated erythrocytes was inhibited by >90% compared to the normal controls (Table 1). The ligand region II binds to its receptor, glycophorin A. Thus, trypsin or neuraminidase treatment of erythrocytes would cleave glycophorin A or remove its sialic acids and eliminate the binding of region II (14).

A comparison of the deduced amino acid sequences of EBA-175 region II for FVO and 3D7 shows that only one residue, at position 286, is differentGlu or Lys, respectively (9). Given that these two strains are essentially identical within region II and yet demonstrate such marked differences in their dependence on sialic acids for invasion, we selected them for the analysis of the capacity of FVO anti-region II antibodies to block merozoite invasion of normal erythrocytes by 3D7 parasites. IgG was purified from the serum of rabbits that had been immunized against FVO EBA-175 region II by a DNA and protein immunization regime. The purified rabbit IgG was adjusted to the original serum volumes and was shown to have similar endpoint dilution titers by enzyme-linked immunosorbent assay (data not shown).

Growth inhibition assays were conducted for two cycles of merozoite invasion in order to enhance the sensitivity of the assay, even though growth inhibition assays using one cycle of invasion demonstrated statistically significant levels of inhibition (data not shown). The results of growth inhibition assays for the FVO and 3D7 strains, conducted in parallel and independently, are shown in Table 2. Essentially, both the FVO and 3D7 strains are inhibited between 28 and 56% at a concentration of 1 mg of IgG/ml (all P values are <0.002). In order to demonstrate that this inhibition was the result of antibodies against region II, soluble recombinant region II protein was added to the parasite cultures to evaluate whether region II protein would absorb the region II antibody and then reverse the level of growth inhibition. The results for a typical experiment are shown in Table 2 and demonstrate that soluble region II protein specifically neutralized the inhibition of region II antibodies by 62 and 77% for FVO and 3D7 strains, respectively, compared to a nonparasite recombinant baculovirus control protein. Analysis of Giemsa-stained thin blood films from one-cycle and two-cycle growth inhibition cultures showed that the morphology of intracellular parasites (trophozoites and schizonts) appeared healthy in the presence of anti-region II antibodies, similar to controls (data not shown). These results indicated that the inhibition of growth was the result of a blockade of merozoite invasion, not interference with schizont maturation or development of immune clusters of merozoites.

The blockade of 3D7 invasion appears to be independent of the parasites' preference for using sialic acid residues on glycophorin A for the invasion process. We attempted to investigate this further by testing whether anti-region II IgG blocked 3D7 parasites from invading neuraminidase-treated erythrocytes. Our results have been varied due to experimental constraints. In an initial experiment, we observed a 42 (P < 0.001) and 25% (P < 0.004) inhibition by anti-region II IgG in normal and neuraminidase-treated erythrocytes, respectively. However, in the case of the neuraminidase-treated erythrocytes, the averages and standard deviations of the final parasitemias with control and test IgGs, were 0.4% ± 0.1% and 0.3% ± 0.1%, respectively, compared to 3.6% ± 0.02% and 2.1% ± 0.06% for normal erythrocytes. At this time we are unaware of human erythrocytes that are completely devoid of sialic acids existing in nature. The capacity of region II antibodies to block the invasion of field isolates that may or may not involve glycophorin A should be studied.

The mechanism by which antibodies against EBA-175 interfere with the sialic acid-dependent invasion pathway of FVO is relatively clear. Rabbit anti-region II antibodies blocked the ligand-receptor interaction between FVO EBA-175 and glycophorin A present on the surface of human erythrocytes (Sim et al., unpublished) and thus could have blocked merozoite invasion by a similar mechanism. The mechanism(s) by which anti-region II antibodies blocked the sialic acid-independent merozoite invasion pathway of 3D7, however, is unclear. P. falciparum 3D7 clearly expresses EBA-175. Rabbit anti-region II antibodies may have interfered stoichiometrically with the ligand-receptor interaction of another protein(s) involved in the alternative invasive pathway (6) in such a way that the ligand-receptor interaction of this alternative pathway was blocked.

We have shown in this study that antibodies against region II not only block invasion involving sialic acids but also block invasion pathways that may not include sialic acids. In addition to region II, some adjacent part of EBA-175 may have a crucial role in merozoite invasion via a pathway that does not involve sialic acids. The fact that EBA-175 has such a universal and yet susceptible role in erythrocyte invasion, in addition to the conservation of EBA-175 region II protein sequence across strains, makes it an attractive therapeutic and vaccine target.