placental malaria is a common complication of pregnancy - associated malaria associated with foetal abortion and stillbirth , and is responsible for low birth weight of the offspring . the factors responsible for the increase in malaria susceptibility during pregnancy , particularly during the first pregnancy , have not been fully elucidated . pregnancy - associated malaria is characterized by the sequestration of plasmodium falciparum - infected erythrocytes in placental intervillous spaces . placental parasites appear to express a specific phenotype defined by the adherence of p. falciparum - infected erythrocytes to the glycosaminoglycans chondroitin sulfate a ( csa ) and hyaluronic acid , expressed by the syncytiotrophoblast . parasite adhesion to csa is mediated by p. falciparum - variant surface antigens ( vsas ) expressed on the surface of infected erythrocytes . placental parasites bind to csa and not to other vsas ligands , such as cd36 and icam-1 , whereas the opposite is true for most parasite isolates from non - pregnant individuals . the expression of this particular vsa by parasites binding to csa in the placenta is thought to be the basis for the higher susceptibility of primigravidae . these women have not been exposed to and have no immunity to such parasites before becoming pregnant . the first pregnancy enables them to develop specific immunity , which protects them during subsequent pregnancies . this hypothesis is supported by the fact that multigravid pregnant women in endemic areas have antibodies to placental parasites acquired during previous malaria - infected pregnancies , and that these antibodies inhibit the cytoadherence of placental parasites to human syncytiotrophoblasts . pregnancy is characterised by a transient depression of cell - mediated immunity of the woman that allows foetal allograft retention , but also interferes with resistance of the mother to various infectious diseases . cellular immune responses to p. falciparum antigens are depressed in pregnant women in comparison with non - pregnant controls . this pregnancy - induced immunosuppression may persist several months after delivery restoration of immune responses to p. falciparum antigens being observed within six months after delivery . in this study , the cellular responses in women at the time of delivery as well as in women 3 months after delivery were assessed . in vitro lymphocyte proliferation and cytokine production were measured for cells obtained from women living in yaounde , cameroon , where malaria transmission is perennial . the study took place in yaounde , cameroon , a rainforest area with hyperendemic p. falciparum malaria and perennial transmission . women delivering at the maternity wards of nkoldongo and djoungolo between june 1998 and january 1999 were enrolled after informed consent was obtained . to aid the identification of women with a placental malaria infection , a thick peripheral blood smear was obtained before delivery and examined for parasites . following identification of an infected individual , the next uninfected woman of same parity presenting to the maternity wards was also enrolled . thick smears of blood from the maternal side of the placenta were examined for the presence of pigment , parasites , or both . another group of women were studied three (  1 ) months after delivery , and 10 ml heparinized venous blood sample was drawn . concanavalin a ( ca ; sigma , st louis , mo ) was used at a final concentration of 10 g / ml . the recall antigen tuberculin purified protein derivative ( ppd ; statens serum institut , copenhagen , denmark ) was used at a final concentration of 12.5 g / ml . the escherichia coli - expressed rmsp119 protein based on p. falciparum merozoite surface protein 1 ( msp-1 ) , was cleaved from a gst - fusion protein and used at a final concentration of 5 g / ml . two p. falciparum lines were used as antigens following in vitro culture . j. gysin , laboratoire de gntique et d'immunologie , marseille , france ) bind to csa and not to other known receptors of p. falciparum   and , consequently , bind to human syncytiotrophoblasts , as do pregnancy - associated parasites . fortnightly , the binding phenotype was selected and checked using csa - coated petri dishes . the unselected w2 p. falciparum line does not bind to csa , nor to human syncytiotrophoblasts ( data not shown ) . infected erythrocytes were obtained from unsynchronised in vitro cultures at a 5 % haematocrit in rpmi 1640 hepes and 10 % heat - inactivated human ab serum . cells were harvested at 5 % parasite density by centrifugation and used to stimulate cultures at 10 % haematocrit . a 100 l aliquot , corresponding to 5.10parasitized erythrocytes , was placed in each well . peripheral blood mononuclear cells ( pbmc ) were isolated on ficoll - paque(pharmacia , uppsala , sweden ) , and cell viability was confirmed by trypan - blue staining . purified pbmc were suspended at 10cells / ml in buffered rpmi 1640 containing 10 % human serum , and 100 l aliquots were placed in triplicate in round bottom 96-well plates . antigens , infected erythrocytes , non - infected erythrocytes ( nie ) , or medium alone were added in 100 l amounts at the indicated concentrations . plates were incubated at 37c in a humidified chamber with 5% co2 for 3 or 6 days . the 3- and 6-day culture supernatants from each triplicate were pooled and stored at -80c . after 6 days , 110 l were removed from culture supernatants and 50 l fresh medium containing 0.5 ci methyl-[h]-thymidine ( specific activity 2 ci / mmole , amersham , les ulis , france ) were added to each well . after a further 16 hr , cells were collected on glass - fiber filters and incorporated radioactivity was measured . stimulation indices ( si ) were calculated by dividing the geometric mean cpm of antigen - stimulated cultures by the geometric mean cpm of unstimulated cultures . a positive response for all antigens il-2 , il-4 , il-10 , and ifn- concentrations were measured in culture supernatants by a sandwich elisa method , using manufacturer 's instructions ( clb amsterdam , the netherlands ) . the detection limit was 5 pg / ml for il-2 , 0.4 pg / ml for il-4 , 3 pg / ml for il-10 , and 2 pg / ml for ifn-. values below this threshold were assigned a value of half the detection limit . for statistical analysis , cytokine concentrations in supernatants of unstimulated cultures or in the presence of nie were subtracted from the values obtained for supernatants of antigen - stimulated cultures , and resulting values were used to define responders and non - responders . differences in prevalence rates between groups were tested by the chi - square test or the chi - square test for trend . differences between groups were analysed by non - parametric methods ( mann - whitney u - test ) . a logistic regression using the maximum likelihood ratio method ( lr procedure of bmdp ) was used to adjust immune responses simultaneously on parity status and infection or age . the study took place in yaounde , cameroon , a rainforest area with hyperendemic p. falciparum malaria and perennial transmission . women delivering at the maternity wards of nkoldongo and djoungolo between june 1998 and january 1999 were enrolled after informed consent was obtained . to aid the identification of women with a placental malaria infection , a thick peripheral blood smear was obtained before delivery and examined for parasites . following identification of an infected individual , the next uninfected woman of same parity presenting to the maternity wards was also enrolled . thick smears of blood from the maternal side of the placenta were examined for the presence of pigment , parasites , or both . another group of women were studied three (  1 ) months after delivery , and 10 ml heparinized venous blood sample was drawn . concanavalin a ( ca ; sigma , st louis , mo ) was used at a final concentration of 10 g / ml . the recall antigen tuberculin purified protein derivative ( ppd ; statens serum institut , copenhagen , denmark ) was used at a final concentration of 12.5 g / ml . the escherichia coli - expressed rmsp119 protein based on p. falciparum merozoite surface protein 1 ( msp-1 ) , was cleaved from a gst - fusion protein and used at a final concentration of 5 g / ml . two p. falciparum lines were used as antigens following in vitro culture . j. gysin , laboratoire de gntique et d'immunologie , marseille , france ) bind to csa and not to other known receptors of p. falciparum   and , consequently , bind to human syncytiotrophoblasts , as do pregnancy - associated parasites . fortnightly , the binding phenotype was selected and checked using csa - coated petri dishes . the unselected w2 p. falciparum line does not bind to csa , nor to human syncytiotrophoblasts ( data not shown ) . infected erythrocytes were obtained from unsynchronised in vitro cultures at a 5 % haematocrit in rpmi 1640 hepes and 10 % heat - inactivated human ab serum . cells were harvested at 5 % parasite density by centrifugation and used to stimulate cultures at 10 % haematocrit . a 100 l aliquot , corresponding to 5.10parasitized erythrocytes , was placed in each well . peripheral blood mononuclear cells ( pbmc ) were isolated on ficoll - paque(pharmacia , uppsala , sweden ) , and cell viability was confirmed by trypan - blue staining . purified pbmc were suspended at 10cells / ml in buffered rpmi 1640 containing 10 % human serum , and 100 l aliquots were placed in triplicate in round bottom 96-well plates . antigens , infected erythrocytes , non - infected erythrocytes ( nie ) , or medium alone were added in 100 l amounts at the indicated concentrations . plates were incubated at 37c in a humidified chamber with 5% co2 for 3 or 6 days . the 3- and 6-day culture supernatants from each triplicate were pooled and stored at -80c . after 6 days , 110 l were removed from culture supernatants and 50 l fresh medium containing 0.5 ci methyl-[h]-thymidine ( specific activity 2 ci / mmole , amersham , les ulis , france ) were added to each well . after a further 16 hr , cells were collected on glass - fiber filters and incorporated radioactivity was measured . stimulation indices ( si ) were calculated by dividing the geometric mean cpm of antigen - stimulated cultures by the geometric mean cpm of unstimulated cultures . a positive response for all antigens il-2 , il-4 , il-10 , and ifn- concentrations were measured in culture supernatants by a sandwich elisa method , using manufacturer 's instructions ( clb amsterdam , the netherlands ) . the detection limit was 5 pg / ml for il-2 , 0.4 pg / ml for il-4 , 3 pg / ml for il-10 , and 2 pg / ml for ifn-. values below this threshold were assigned a value of half the detection limit . for statistical analysis , cytokine concentrations in supernatants of unstimulated cultures or in the presence of nie were subtracted from the values obtained for supernatants of antigen - stimulated cultures , and resulting values were used to define responders and non - responders . differences in prevalence rates between groups were tested by the chi - square test or the chi - square test for trend . differences between groups were analysed by non - parametric methods ( mann - whitney u - test ) . a logistic regression using the maximum likelihood ratio method ( lr procedure of bmdp ) was used to adjust immune responses simultaneously on parity status and infection or age . a total of 47 women were enrolled at delivery , and 41 ( including 15 of these ) were investigated 3 months after delivery . among the former , 17 were primigravidae ( mean age : 18.8  1.7 years ; 7 having presented with a placental infection ) and 30 multigravidae ( mean age : 26.1  4.2 years ; 15 having presented with a placental infection ) . among the latter , 17 were primigravidae ( mean age : 20.8  3.6 years ; 9 having presented with a placental infection ) and 24 multigravidae ( mean age : 26.1  5.0 years ; 12 having presented with a placental infection ) . the two groups of women investigated at delivery and 3 months after delivery were similar , as regards to age , parity , area of residence , and season of bleeding ( all p > 0.2 ) . in the presence of concanavalin a and ppd , lymphocytes from most women proliferated whether these cells were collected at delivery or 3 months later . both the prevalence rates and the mean levels in the proliferation assay were higher 3 months after delivery than at delivery ( table 1 ) . the prevalence rate of women whose cells proliferated in response to msp-1 or rp5 , but not to w2 , was also significantly higher 3 months after delivery than at delivery . similarly , the mean level of responses was higher . no differences were found on proliferation responses at delivery according to parity and malaria status ( all p > 0.1 ) , whereas these differences in response to rp5 after delivery appeared to be related to both the occurrence of a p. falciparum infection of the placenta , and to the parity status of the woman . three months after delivery , cell proliferation in response to rp5 was observed in 53 % of the mothers who had a parasite - positive placenta at delivery , while cells from only 17 % of the other women proliferated ( p = 0.02 ) ( see fig . the mean levels of responses to rp5 were also increased in women who had presented with a malarial infection of the placenta three months earlier ( 2.4 ( 1.4  4.0 ) vs. 1.4 ( 1.0  1.9 ) , mann - whitney u - test , p = 0.07 ) . likewise , cells from 50 % of multiparous women proliferated in response to rp5 , while those of only 18 % of primiparous proliferated ( p = 0.04 ) ( fig . this difference was also observed for the mean level of response , which was higher in multiparous women ( 2.3 ( 1.4  3.7 ) vs. 1.4 ( 1.0  2.0 ) , mann - whitney u - test , p = 0.08 ) . logistic regression demonstrated that both placental infection ( p = 0.03 ) and parity ( p = 0.05 ) were independent predictors of the proliferative response to rp5 . no such difference was observed with either the msp-1 antigen or the w2 p. falciparum strain . in vitro proliferative ( lpa ) and cytokine prevalence rates of response of peripheral blood mononuclear cells from cameroonian women 3 months after delivery . panel a , women having presented ( n = 20 , white ) or not ( n = 21 , black ) with a p. falciparum infection of the placenta ; panel b , primiparae ( n = 17 , black ) and multiparae ( n = 24 , white ) . cona : concanavalin a ; ppd : tuberculin purified protein derivative ; msp1 : recombinant protein from p. falciparum merozoite surface protein-1 ; rp5 and w2 : erythrocytes infected with the rp5 and w2 p. falciparum lines , respectively . in vitro proliferative responses of peripheral blood mononuclear cells collected at delivery and 3 months later from cameroonian women . data are expressed as prevalence rates and geometric means ( 95 % confidence interval ) of stimulation indices . p value of the difference in prevalence rates , chitest p value of the difference in mean levels , mann - whitney u - test the in vitro production of ifn- , il-2 , il-10 , and il-4 was investigated three months after delivery and compared according to parity and to placenta malaria status at delivery . no difference in cytokine response was observed with respect to placenta malarial status at delivery ( i. e. with or without placental infection ) ( fig . however , considering the parity status ( primiparous versus multiparous ) differences were observed in cytokine production , especially in response to rp5 ( fig . the prevalence rates of il-2 production were increased in multiparous women ( all p = 0.05 ) ( fig . 1b ) in response to the three malaria antigens , while no differences were observed with concanavalin a or ppd . the prevalence of production of il-2 and il-4 by cells from multiparous women was twice as high ( both p < 0.02 ) , and a similar trend was observed for ifn- and il-10 , although the differences did not reach significance ( p = 0.16 and 0.08 respectively ) . moreover , the prevalence rate of responses to rp5 gradually increased with parity ( fig . 2 ) , this increase being significant for lymphocyte proliferation and il2 and il4 production ( chitest for trend , p = 0.01 , 0.002 and 0.05 , respectively ) . such parity - related changes were not observed with the other malarial antigens . among multiparous women , except in the case of il2 production in presence of w2 , these responses did not increase with parity ( fig . 2 ) . the prevalence rates of il-10 response to msp-1 decreased with parity ( p = 0.04 ) . in vitro proliferative and cytokine prevalence rates of response to msp1 ( recombinant protein from p. falciparum merozoite surface protein-1 ) , or to erythrocytes infected with the rp5 or the w2 p. falciparum strain from peripheral blood mononuclear cells collected 3 months after first ( n = 17 , white ) , second ( n = 9 , grey ) , third ( n = 7 , stripped ) , or = fourth ( n = 8 , black ) delivery in cameroonian women . the geometric mean levels of production of all cytokines in response to concanavalin a , as well as those of ifn- and il-2 in response to ppd were 10 times higher than responses to the three malaria antigens ( fig . the level of production of these cytokines in response to rp5 was also increased also two to six times higher in cells from multiparous women , and the difference was significant for il-2 and il-4 ( 5.1 vs 1.5 pg / ml , 5.3 vs 0.5 pg / ml respectively ; all p < 0.04 ) . in contrast , the responses to the other antigens were only moderately affected by the parity status . il2 production in the presence of rp5 or w2 also increased with age in an univariate analysis , but logistic regression demonstrated that parity remained the only independent predictor of the il2 response to rp5 ( p = 0.02 ) and w2 ( p = 0.005 ) . in vitro cytokine production in response to con a : concanavalin a ; ppd : tuberculin purified protein derivative ; msp1 : recombinant protein from p. falciparum merozoite surface protein-1 ; rp5 and w2 : erythrocytes infected with the rp5 and w2 p. falciparum strains , respectively from peripheral blood mononuclear cells collected 3 months after delivery in cameroonian women . pregnancy is an immunological balancing act in which the mother 's immune system has to remain tolerant to the foetus and yet maintain immune competence for defense against microorganisms . several studies have documented a decrease of cell - mediated immune responses to malaria parasite antigens during pregnancy   and early post - partum   in association with increased susceptibility to malaria . consequently , the exploration of p. falciparum specific immune responses performed during this time is unlikely to produce results that are easy to interpret . our rationale for not investigating cytokine secretion at delivery , but for performing full analysis of the cellular immune response three months after delivery , was that we expected that immunosuppression would be abolished by this time . indeed , we confirmed an important systemic lowering of the malaria - specific and non - specific cellular responses , as the proliferative response of peripheral blood mononuclear cells was highly reduced at delivery , as compared to three months later . regarding the relationship between parity status and cellular responses to the three p. falciparum antigens used , different profiles were observed . il2 prevalence rate in response to all malaria antigens gradually increased with the number of previous parities , independent of age , suggesting the construction of t cell memory during successive pregnancies . in response to the rp5 line , the prevalence rate and the level of response increased with the number of previous parities in most of the assays used . this relationship was only evident for il2 in responses to the other p. falciparum line or a recombinant protein based on msp-1 , an antigen shared by all p. falciparum lines . this demonstrates the occurrence of an immune response that is more specific for a parasite phenotype expressed by the rp5 line in women who have had repeated pregnancies . the rp5 line of p. falciparum expresses the specific cytoadherence properties of the pregnancy - associated parasites , whereas the w2 line does not cytoadhere to syncytiotrophoblast ( maubert , unpublished data ) . however , p. falciparum lines might not be the most appropriate tool to dissect the immune response , as numerous antigens are implicated . pregnancy - associated parasites adhere to csa expressed on the placental endothelium , representing a phenotype that is not shared with parasites isolated from non - pregnant hosts , but that is expressed by the rp5 line . several molecules on the surface of infected erythrocytes suggested as acting as counter - receptors for these epithelial molecules and the major adherence receptor appears to be pfemp-1 . pfemp-1 molecules are highly antigenic and are encoded by a diverse multigene family , and expression on the surface of erythrocytes can switch during the course of a clonal infection . the hypothesis   that the strains of p. falciparum implicated in placental malaria are representative of a unique sub - population , was recently supported by the demonstration that the expressed pfemp-1 in infected placental parasite samples are closely related to each other   among pregnant women , primiparous women with a non - infected placenta have the highest likelihood of never having been infected with pregnancy - associated parasites . however , even in these women , an infection may have occurred during early pregnancy , explaining why some of them had raised responses to the rp5 strain after delivery . the higher proportion of multiparous women that responded to the rp5 strain probably reflects a pregnancy - associated parasite - specific immunization during a previous parasitized pregnancy . this likelihood of having been infected , as well as the proportion of responders , increases with parity . in line with our data , a previous report demonstrated that the acquisition of antibodies against the rp5 line and against csa - adhering parasites correlated with the number of past pregnancies . moreover , antibodies directed against csa - adhering parasites and acquired during an infected pregnancy inhibited the cytoadherence of csa - adhering parasites to human syncytiotrophoblasts . it is likely that women having multiple pregnancies , who develop an immune response to rp5 , are developing immunity to pregnancy - associated parasites , thereby decreasing the risk of pregnancy - associated malaria during subsequent pregnancies . proposed a model in which re - circulation of memory t lymphocytes from the intervillous blood to local lymphoid tissue facilitates the maintenance of local immunity . both cellular and humoral components may account for the lower susceptibility of multigravidae to malaria . understanding the mechanisms involved in protection of multigravidae to placental malaria will have consequences in public health management , both in terms of drug - based control measures and malaria vaccines . indeed , to limit costs , some countries advise restricting drug prophylaxis against malaria during pregnancy to the primiparae , the most sensitive group of women . as the lower susceptibility of multiparae may rely on an earlier exposure and immunization against placental parasites , a total prevention of malaria during the first pregnancy may inhibit the development of immune responses against pregnancy - associated parasites . however , no difference in susceptibility to malaria was observed during the second pregnancy of women who had or had not received drug prophylaxis during their first pregnancy . this is probably because , in areas in which malaria is endemic , prophylaxis is rarely taken before the fifth or sixth month of pregnancy , and its efficacy is frequently impaired by drug resistance . it is likely that drug prophylaxis will limit placental colonization , without inhibiting the development of specific immunity . bull and colleagues reported that protection against disease is dependent on a variant - specific immune response directed against vsas that probably include members of the pfemp-1 family . this supports the idea that pfemp-1 could be used to develop an anti - disease vaccine , although the large size of the pfemp-1 family raises doubts about the feasibility of this approach . in the case of pregnancy - associated malaria , the number of antigenic variants may be very small , and therefore pfemp-1 may be a good vaccine candidate for pregnancy - associated malaria . further studies using specific antigen from placental malaria parasites will provide additional information about the development of both cellular and humoral immunity in pregnant women . author : 1 n f conceived of the study , carried out the studies , the immunology tests , the immunoassays and the statistical analysis and drafted the manuscript . author 4 : m m participated in the immunoassays author 5 : i s produced the recombinant protein preparation author 6 : mc participated in the design of the study and statistical analysis author 7 : a a h produced the recombinant protein preparation author 8 : gc participated in the design of the study author 9 : pd participated in the design of the study , statistical analysis , drafted the manuscript and coordinated the study . this work was supported by ird , aupelf / uref , inserm , and eu contracts ts3ct94 0272 and qlk2-ct-1999 - 01293 . b. m. was the recipient of a fellowship from the french ministre de l'education nationale , de l'enseignement suprieur et de la recherche . we acknowledge the strong support of the staff of the maternity clinics of nkolndongo and of the djoungolo hospital , who were in charge of the collection of biological samples and other data . we are also grateful to andr garcia for help in statistical analysis , and to christian boudin for his support and scientific advice .
backgroundmalaria in pregnancy is characterised by the sequestration of plasmodium falciparum - infected erythrocytes in placental intervillous spaces . 
 placental parasites express a specific phenotype , which allows them to cytoadhere to chondroitin sulfate a expressed by syncytiotrophoblasts . 
 malaria infection during pregnancy allows the acquisition of antibodies against placental parasites , these antibodies are thought to be involved in protection during subsequent pregnancies.methodsto investigate the development of a cellular response to placental parasites during pregnancy , peripheral blood mononuclear cells were collected from women at the time of their confinement . 
 the study was performed in cameroon where malaria transmission is perennial . in vitro cell proliferation and cytokine production 
 were measured in response to non - malarial activators ( concanavalin a and ppd ) , a recombinant protein from p. falciparum msp-1 , and erythrocytes infected by two p. falciparum lines , rp5 and w2 . like placental parasites , the rp5 line , but not w2 , adheres to chondroitin sulfate a and to syncytiotrophoblasts.resultsthe proliferative response to all antigens was lower for cells obtained at delivery than 3 months later . 
 most interestingly , the cellular response to the rp5 line of p. falciparum was closely related to parity . 
 the prevalence rate and the levels of response gradually increased with the number of previous pregnancies . 
 no such relationship was observed with w2 line , or msp-1 antigen.conclusionsthis suggests the occurrence of an immune response more specific for the rp5 line in women having had multiple pregnancies , and who are likely to develop immunity to pregnancy - associated parasites . 
 both humoral and cellular mechanisms may account for the lower susceptibility of multigravidae to malaria .