[PMC free article] [PubMed] [Google Scholar] 6. and in 76.9% of secundigravidae (= 0.02). The antibodies against CSA-binding parasites inhibited the cytoadherence of a CSA-adherent parasite strain to the human placental trophoblast. Our data support the idea that the higher susceptibility of primiparae is related to a lack of a specific immune response to placental parasites. genome is able to encode around one hundred PfEMP1 molecules, these molecules are expressed not simultaneously but one at a time. In addition to their antigenic properties, PfEMP1 molecules mediate the cytoadherence of infected erythrocytes to a variety of endothelium cells that express receptors. Receptors potentially bound by strain that binds only to CSA (and was therefore used to mimic placental parasites). We next described the acquisition of immunity against pregnancy-associated parasites (PAPs) in women longitudinally monitored in Ebolowa, Cameroon, during their first two pregnancies. Finally we present evidence that antibodies directed against PAPs acquired during the first infected pregnancy inhibit the cytoadherence of placental parasites to the human syncytiotrophoblast and may account for the lower frequency of malaria in multigravidae. MATERIALS AND METHODS Samples from Yaound. In this study, we enrolled all women delivering babies in the maternity wards of Nkolndongo, Yaound, Cameroon, from June 1996 to April Vps34-IN-2 1997, after they gave their oral informed consent. Women delivering during weekends were excluded. After the women had delivered, blood samples were taken by puncture and plasma was frozen. A crush smear was made from an excised piece of placenta. Placental blood thick films were air dried, Giemsa stained, read by microscopy over 50 fields at a 1,000 magnification, and considered positive when parasites or malarial pigments were observed. Peripheral blood parasites were cryopreserved. Nonpregnant subjects (women and Vps34-IN-2 men) were recruited in the dispensaries of Nkolndongo and Messa, in the same town. Plasma samples from all participants were frozen, and parasites, if any were isolated, were cryopreserved. Serum samples from Ebolowa. To study the evolution of line Plxnc1 (a gift from J. Gysin, Laboratoire de Gntique et dImmunologie, IMTSSA, Parc du Pharo, Marseille, France) binds to CSA and not to the other known receptors of (8) and consequently binds to the human syncytiotrophoblast (14). In our laboratory, the binding phenotype was maintained by a fortnight flotation on plasmagel (18). Three parasite isolates from pregnant women, four from nonpregnant women, and the RP5 strain were thawed and cultivated in candle jars according to standard procedures (21) at a 5% hematocrit with 10% heat-inactivated human AB serum added to RPMI 1640-HEPES (25 mM). All tests were performed when parasites were in the late stage (from late trophozoite to young schizont). Parasites from isolates were used during the first life cycle. Agglutination test. Serum antibodies to infected erythrocytes (IEs) were detected by a modification of the antibody-mediated agglutination assay (11). Serum (2.5 l) was deposited in a 96-well microtitration plate (U bottom). A parasite culture at the mature stage was washed and resuspended in phosphate-buffered saline, pH 7.4, at an 11% hematocrit, and 22.5 l of this suspension containing 0.01% acridine orange was added into each well (final hematocrit, 10%; final serum concentration, 10%). After a 90-min rotation at room temperature on a Coulter mixer (a 45 inclination on a 22-round-per-minute rotating dish), 50 l of phosphate-buffered saline was added and 20 l of the suspension was examined between an examination slide and a 22- by 22-mm cover slide. Agglutinates were examined under UV and bright-field illumination. The assay result was considered positive when at least five agglutinates of at least three IEs were counted, and the result was quantified by the geometric mean of the five biggest agglutinates. Inhibition of the cytoadherence to human trophoblast by immune-phase sera. The effect of sera on the cytoadherence of the RP5 strain was assessed by using a modification of the cytoadherence assay previously described (14). Briefly, cytotrophoblasts were purified from a human placenta by negative immunoselection for CD9 (24). Cells were seeded in microwells on plastic dishes coated with parafilm (15) and cultivated for 7 to 15 days before the cytoadherence assay was performed. The RP5 strain was used Vps34-IN-2 when presenting with a majority of mature stages. Parasite culture at a 10% hematocrit was.