However, it is unresolved whether this finding reflected the presence of cross-reactive antibodies to conserved epitopes or, alternatively, the presence of antibodies with multiple specificities in individual serum samples. vaccination with a single domain or isolate might be hindered by antigenic diversity. Although immunity to malaria may exist before pregnancy, malaria is more prevalent and severe during pregnancy, especially among primigravid women. infection is characterized by the accumulation in the placenta of mature-stage parasite-infected erythrocytes (IEs) [1, 2], mediated through adhesion to chondroitin sulfate A (CSA) [3, 4], and other molecules, such as hyaluronic acid (HA) and nonimmune immunoglobulins [5-8]. Early in a first pregnancy, women generally lack antibodies to placental-binding IEs, which suggests that these Pantoprazole (Protonix) parasites represent novel variant antigens to which women have not been exposed previously [4, 9-11]. Reduced susceptibility is observed in women who have had several pregnancies exposed to malaria, because of the acquisition of specific immunity [12]. Antibodies to surface antigens expressed by placental isolates and isolates that adhere to CSA or both CSA and HA are acquired after exposure to placental malaria [4, 9-11, 13, 14]. These antibodies generally are more prevalent in multigravidae than in primigravidae [4, 9-11], corresponding Pantoprazole (Protonix) with a reduced risk of malaria during pregnancy, and, in cross-sectional studies, there is some association between these antibodies and improved pregnancy outcomes among women at delivery [15, 16]. The major target of antibodies to antigens expressed on the surface of IEs is the highly diverse erythrocyte membrane protein 1 (PfEMP1), which is encoded by the multigene family [17-19]. Antigenic variation of PfEMP1, through switching expression of different genes, facilitates evasion of host immune responses, and specific variants of PfEMP1 mediate adhesion to CSA [20, 21]. Recently, proteins [23]. Together, these findings suggest that PfEMP1 is an important target of acquired and possibly protective antibodies. The degree of antigenic diversity or conservation of antigens expressed by placental IEs is currently unclear. Serum samples obtained from women in different geographic regions inhibited adhesion to CSA of the same placental isolates, suggesting the expression of conserved antigens [9]. However, it is unresolved whether this finding reflected the presence of cross-reactive antibodies to conserved epitopes or, alternatively, the presence of antibodies with multiple specificities in individual serum samples. In contrast, in a separate study, agglutinating antibodies to antigens expressed on the surface of placental IEs were found to be variant specific to a significant extent [4]. In studies of children and nonpregnant adults, the presence and significance of cross-reactive antibodies to IE surface antigens have, in general, been controversial [25]. Here, we have investigated these issues by examining antigenic diversity and differences among defined CSA-binding and CSA-HA-binding isolates known to be expressing as the dominant gene, by comparing defined CSA-binding isolates and IEs harvested from infected placentas and by examining whether acquired antibodies comprise variant-specific and/or cross-reactive antibodies to placental IEs. SUBJECTS, MATERIALS, AND METHODS P. falciparum was cultured in medium supplemented with either 10% vol/vol pooled human serum (obtained from donors who were residents of Australia) or 5% serum and 0.25% Albumax II (Gibco), as described elsewhere [4]. isolates CS2, HCS3, and 3D7-CSA are genetically distinct and were generated by selection for adhesion to CSA, Pantoprazole (Protonix) as described elsewhere [8]. The specificity of adhesion to CSA and HA has been established elsewhere [5, 8, 26]. Isolate E8B adheres to CD36 and intercellular adhesion molecule 1 and is isogenic to CS2 [8]. Clonality and genetic identity were determined by polymerase chain reaction analysis of and alleles, by use HNRNPA1L2 of established methods [27]. Cultures were free of species, as determined by polymerase chain reaction. Placental isolates were recovered from pregnant women attending the labor ward of the Queen Elizabeth Central Hospital in Blantyre, Malawi [4]. Written, informed Pantoprazole (Protonix) consent was obtained from all women who participated in the study. IEs (predominantly mature trophozoites) were harvested from freshly delivered infected placentas, as described elsewhere [4]. Parasite adhesion and inhibition assays Adhesion assays and assays to measure the inhibition of adhesion by serum and/or plasma were performed, as described elsewhere [4,.