[PMC free article] [PubMed] [Google Scholar] 3. and yet susceptible role in erythrocyte invasion clearly supports its inclusion in a multivalent malaria vaccine. The need for an effective malaria vaccine or additional therapies against the human malaria agent 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 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 and (1, 2). Region II appeared to be conserved across 16 different 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 1G244 or neuraminidase-treated human erythrocytes devoid of sialic acids, generally correlated closely with the ability of these erythrocytes to be invaded by (4, 11). However, for some 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 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 MkMk 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 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 1G244 strains, which have the capability to invade erythrocytes by distinct pathways, were similarly blocked by antibodies against EBA-175 region II. MATERIALS AND METHODS Parasites. Cloned 3D7 (human challenge strain) and FVO (Vietnam 1G244 isolate adapted to Aotus monkeys) strains of were cultured and synchronized by temperature cycling through 37, 40, and 17C (8). Schizont-infected erythrocytes were Percoll purified for analysis of merozoite invasion of enzymatically treated erythrocytes. Erythrocytes and enzyme pretreatments. Human Lum blood was collected in a 10% (final concentration) 1G244 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 4C. Erythrocytes were washed and treated with 0.2 U of neuraminidase (Gibco BRL, Gaithersburg, Md.) per 109 erythrocytes as previously described (5) or were treated with 1 mg of trypsin (Sigma, St. Louis, Mo.) 1G244 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..

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