A Protective Glycosylphosphatidylinositol-Anchored Membrane Protein of Plasmodium yoelii Trophozoites and Merozoites Contains Two Epidermal Growth Factor-Like Domains

Efforts to develop an effective malaria vaccine are concentrated on Plasmodium falciparum, the major cause of malaria morbidity and mortality worldwide (45). Several promising vaccine targets have been identified in each of the major developmental stages of this protozoan parasite (24). These P. falciparum vaccine candidate antigens have been largely identified based on their functional importance and/or as targets of human immune responses. Additional in vivo studies involving the rodent malaria parasites Plasmodium yoelii and Plasmodium chabaudi have facilitated efforts to assess the vaccine potential of several antigens of interest (3, 5, 9, 11, 14–16, 26, 28, 30, 34, 42). Evaluation of the most promising candidate antigens has progressed to human clinical trials, and the initial results are encouraging (18). Even so, it is clear that further efforts to improve overall malaria vaccine efficacy are needed. This includes the characterization of additional parasite antigens and epitopes that induce protective responses upon immunization. Blood-stage malaria vaccine studies have emphasized the mapping of B-cell epitopes of plasmodial antigens that are targets of protective antibodies. Many immunodominant, linear B-cell epitopes have been identified in association with domains of proteins that contain repeated blocks of amino acids. Many of these determinants are polymorphic and have been suggested to be part of an immune evasion strategy (31, 37). On the other hand, P. falciparum repeat domains, such as those found in the circumsporozoite protein, the ring-infected erythrocyte surface antigen, the serine repeat antigen, and merozoite surface protein 2 (MSP-2) may in fact be targets of protective antibodies (27). A second, very different group of potentially protective B-cell epitopes has been mapped to domains of malaria antigens that are conformationally constrained by multiple disulfide bonds. In P. falciparum, these include the cytoadherence domains of erythrocyte membrane protein 1 (4), the ectodomain of apical membrane antigen 1 (3, 14, 23, 44), the binding domain of the 175-kDa erythrocyte binding antigen (1, 32), and as a group, the epidermal growth factor (EGF)-like domains of MSP-1, MSP-4, MSP-5, and the sexual-stage antigens Pfs25 and Pfs28 (5, 11, 15, 17, 25, 29, 30, 35, 36). Generally these epitopes are less variable between P. falciparum strains, exhibit conservation across species, and appear to be functionally important. As such, they are of considerable interest for malaria vaccine development. Previously, we reported that immunization with a particulate fraction of blood-stage antigens (pAg) can protect mice against P. yoelii malaria (10). We attempted to utilize the responses of these immunized and protected animals to identify novel, protective blood-stage antigens. One such antigen, pypAg-1, is a novel membrane protein of P. yoelii-infected erythrocytes that was shown to express at least two distinct protective epitopes (8). Using this immunization-based approach to antigen discovery, we now report the identification and characterization of pypAg-2, a second novel antigen of P. yoelii. We present data indicating that pypAg-2 is a glycosylphosphatidylinositol (GPI)-anchored membrane protein of trophozoites and merozoites that lacks repetitive amino acid sequences but contains two C-terminal EGF-like domains. Most importantly, we report that immunization with recombinant pypAg-2 protects mice against lethal P. yoelii blood-stage malaria.