|High avidity antibodies to full-length VAR2CSA correlate with absence of placental malaria.|
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|PMID: 22761948 Owner: NLM Status: MEDLINE|
|VAR2CSA mediates sequestration of Plasmodium falciparum-infected erythrocytes in the placenta, increasing the risk of poor pregnancy outcomes. Naturally acquired antibodies (Ab) to placental parasites at delivery have been associated with improved pregnancy outcomes, but Ab levels and how early in pregnancy Ab must be present in order to eliminate placental parasites before delivery remains unknown. Antibodies to individual Duffy-binding like domains of VAR2CSA have been studied, but the domains lack many of the conformational epitopes present in full-length VAR2CSA (FV2). Thus, the purpose of this study was to describe the acquisition of Ab to FV2 in women residing in high and low transmission areas and determine how Ab levels during pregnancy correlate with clearance of placental parasites. Plasma samples collected monthly throughout pregnancy from pregnant women living in high and low transmission areas in Cameroon were evaluated for Ab to FV2 and the proportion of high avidity Ab (i.e., Ab that remain bound in the presence of 3M NH(4)SCN) was assessed. Ab levels and proportion of high avidity Ab were compared between women with placental malaria (PM(+)) and those without (PM(-)) at delivery. Results showed that PM(-) women had significantly higher Ab levels (p = 0.0047) and proportion of high avidity Ab (p = 0.0009) than PM(+) women throughout pregnancy. Specifically, women with moderate to high Ab levels (>5,000 MFI) and those with ≥ 35% high avidity Ab at 5-6 months were found to have 2.3 (95% CI, 1.0-4.9) and 7.6-fold (p = 0.0013, 95% CI: 1.2-50.0) reduced risk of placental malaria, respectively. These data show that high levels of Ab to FV2, particularly those with high avidity for FV2, produced by mid-pregnancy are important in clearing parasites from the placenta. Both high Ab levels and proportion of high avidity Ab to FV2 may serve as correlates of protection for assessing immunity against placental malaria.|
|Yeung Lo Tutterrow; Ali Salanti; Marion Avril; Joseph D Smith; Ian S Pagano; Simon Ako; Josephine Fogako; Rose G F Leke; Diane Wallace Taylor|
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|Type: Journal Article; Research Support, N.I.H., Extramural; Research Support, N.I.H., Intramural; Research Support, Non-U.S. Gov't Date: 2012-06-26|
|Title: PloS one Volume: 7 ISSN: 1932-6203 ISO Abbreviation: PLoS ONE Publication Date: 2012|
|Created Date: 2012-07-04 Completed Date: 2013-01-04 Revised Date: 2014-06-03|
Medline Journal Info:
|Nlm Unique ID: 101285081 Medline TA: PLoS One Country: United States|
|Languages: eng Pagination: e40049 Citation Subset: IM|
|APA/MLA Format Download EndNote Download BibTex|
Antigens, Protozoan / immunology*
Malaria, Falciparum / complications, immunology*
Pregnancy Complications, Parasitic / immunology*
|R01AI071160/AI/NIAID NIH HHS; U19 AI089688/AI/NIAID NIH HHS; U19AI089688/AI/NIAID NIH HHS; UO1AI43888/AI/NIAID NIH HHS|
|0/Antigens, Protozoan; 0/VAR2CSA protein, Plasmodium falciparum|
Journal ID (nlm-ta): PLoS One
Journal ID (iso-abbrev): PLoS ONE
Journal ID (publisher-id): plos
Journal ID (pmc): plosone
Publisher: Public Library of Science, San Francisco, USA
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Received Day: 9 Month: 4 Year: 2012
Accepted Day: 31 Month: 5 Year: 2012
collection publication date: Year: 2012
Electronic publication date: Day: 26 Month: 6 Year: 2012
Volume: 7 Issue: 6
E-location ID: e40049
PubMed Id: 22761948
Publisher Id: PONE-D-12-09930
|High Avidity Antibodies to Full-Length VAR2CSA Correlate with Absence of Placental Malaria Alternate Title:Antibodies to Full-Length VAR2CSA|
|Yeung Lo Tutterrow1|
|Joseph D. Smith3|
|Ian S. Pagano4|
|Rose G. F. Leke5|
|Diane Wallace Taylor1*|
|Henk D. F. H. Schalligedit1||
1Department of Tropical Medicine, Medical Microbiology and Pharmacology, John A. Burns School of Medicine, University of Hawai'i-Mānoa, Honolulu, Hawaii, United States of America
2Department of International Health, Immunology and Microbiology, Centre for Medical Parasitology, University of Copenhagen and Department of Infectious Diseases, Copenhagen University Hospital (Rigshospitalet), Copenhagen, Denmark
3Seattle Biomedical Research Institute, Seattle, Washington, United States of America
4Cancer Research Center, University of Hawaii-Mānoa, Honolulu, Hawaii, United States of America
5The Biotechnology Center, University of Yaoundé I, Yaoundé, Cameroon
|Royal Tropical Institute, The Netherlands
|Correspondence: * E-mail: firstname.lastname@example.org
Contributed by footnote: Conceived and designed the experiments: YLT AS MA JDS RGFL DWT. Performed the experiments: YLT MA ISP SA JF. Analyzed the data: YLT MA ISP. Contributed reagents/materials/analysis tools: AS JDS RGFL. Wrote the paper: YLT AS MA JDS RGFL DWT.
Plasmodium falciparum infections increase the risk of maternal anemia, delivery-related complications and poor pregnancy outcomes –. A major reason for the severity of malaria during pregnancy is the binding of infected erythrocytes (IE) via VAR2CSA to chondroitin sulfate A (CSA) on syncytiotrophoblasts and extracellular matrix in the intervillous space of the placenta –. As a result, IE sequester in the placenta causing placental malaria (PM). After one or more pregnancies, women develop antibodies (Ab) to VAR2CSA that improve pregnancy outcomes, including reduced risk of maternal anemia , , low birth weight babies , , and placental parasitemia . In vitro and ex vivo studies show that naturally acquired Ab block IE from binding to placenta tissue and CSA – and promote phagocytosis by macrophages –; however, characteristics of the Ab response required for women to clear placental parasites remains unclear. It is also unknown how development of Ab to VAR2CSA differs between women living in high versus low transmission areas. Answers to these questions will provide crucial information for the forthcoming VAR2CSA clinical trials and for assays to evaluate protection from placental malaria.
In this study, the acquisition of Ab specific for full-length VAR2CSA (FV2) was determined using plasma collected longitudinally from women living in areas of high and low perennial malaria transmission, prior to implementation of intermittent presumptive treatment and insecticide-treated bednets. This is the first study to report the natural acquisition of Ab to all Duffy-binding like (DBL) domains, as previous studies have measured either the binding of Ab to the surface of IE and/or to recombinant DBL domains of VAR2CSA –. It is important to examine naturally acquired Ab to FV2, since individual DBL domains exhibit lower specificity and binding affinity for CSA compared to full-length VAR2CSA , ; while, measuring Ab to the surface of IE is less specific compared to Ab responses to recombinant FV2.
In previous studies, an association between inhibition of binding and clearance of parasites from the intervillous space of the placenta was not found using serum samples collected at delivery , . Here we sought to determine if Ab levels at the end of the first, during the second, and/or third trimesters correlated with clearance of placental infections by comparing the responses of women who had placental malaria (PM+) and those without (PM−) at delivery. Furthermore, since immune exposure to VAR2CSA is primarily pregnancy-associated, the restricted exposure raises the questions of whether and how soon women produce high avidity Ab to VAR2CSA (i.e., Ab with strong-binding to FV2). High avidity Ab resulting from affinity maturation are often correlated with strong activities against viruses ,  and bacteria , as well as protection from diseases . It was recently reported that individuals residing in malaria endemic areas with high affinity Ab to P. falciparum merozoite surface protein-2 (MSP-2) had prolonged periods without clinical malaria . Accordingly, we also investigated the importance of high avidity anti-VAR2CSA Ab in clearing parasites of the placental phenotype.
The composition of women in Ngali II (n = 39) and Yaoundé (n = 50) included in this study were similar with respect to age, length of pregnancy, and proportion of primigravidae (Table 1). Significantly more women in Ngali II became slide-positive during pregnancy (p = 0.01), but the prevalence of slide-positivity did not differ significantly between the two sites when analyzed by trimesters.
For ease, Ab responses were divided into four categories: negative (below cut-off), low (above cut-off, but <5,000 MFI), intermediate (5,000–10,000 MFI) and high (>10,000 MFI). Women with intermediate to high Ab levels were considered to have strong Ab responses. Cut-off values were defined by the mean+2 SD from corresponding endemic male controls (Fig. 1e–f). Acquisition of Ab to FV2 differed in women living in Ngali II and Yaoundé (Fig. 1). At 3 months of pregnancy, 28% of primigravidae in Ngali II had Ab (Fig. 1a); whereas, none of the primigravidae in Yaoundé were Ab-positive (Fig. 1b). By 4 months, 20% of primigravidae in Ngali II had developed strong responses (i.e., MFI >5,000); whereas, almost none of the primigravidae in Yaoundé developed strong responses prior to delivery. Likewise, multigravidae in Ngali II had more rapid responses than multigravidae in Yaoundé (Fig. 1c – d) and a higher proportion of multigravidae in Ngali II produced strong Ab responses at 6 (p<0.0001) and 8 months (p = 0.0006) than multigravidae in Yaoundé. Clearly, malaria transmission strongly influenced the acquisition of Ab to FV2.
The response to FV2 was predominantly pregnancy-associated as adult males living in two sites had very low reactivity with FV2 (Fig. 1e–f). Only 1 of 40 (2.5%) males had intermediate Ab levels and 6/40 (15%) had low levels at both sites. Thus, it is likely that most pregnant women with low Ab to FV2 seroconverted during pregnancy.
Data on placental malaria status at delivery were available for 27/39 women in Ngali II and 48/50 in Yaoundé. Characteristics of this subgroup of women did not differ from those in Table 1. Ab levels throughout the course of pregnancy were compared between PM+ and PM− women (Fig. 2) using multilevel polynomial regression analysis. In Ngali II, Ab levels increased in a liner fashion (p = 0.0003) in both PM− and PM+ women with both groups having similar Ab levels by delivery (Fig. 2a). Notably, PM− women had significantly higher Ab levels throughout the course of pregnancy than PM+ women (p = 0.005). Since all PM− women were multigravidae, adjustment could not be made for gravidity in the analysis across groups. However, within the PM+ group, there was no significant difference in Ab levels to FV2 between primigravidae and multigravidae (p = 0.54). Therefore, the absence of PM at delivery in Ngali II correlated with Ab levels over time.
In Yaoundé, 23/48 women were PM− and failed to make Ab to FV2 (data not shown). These women had detectable submicroscopic infections or very low parasitemia by microscopy, but they did not produce Ab to FV2. Data from the remaining 25 women who were seropositive for FV2 showed a similar trend to that found in women living in Ngali II. PM− women in Yaoundé had consistently higher levels of Ab compared to PM+ women, although the difference in this small group of women did not reach statistical significance (Fig. 2b). Overall, the magnitude of Ab response was lower in women living in Yaoundé.
Placental malaria-negative women at both study sites were exclusively multigravidae (i.e., ≥2 pregnancies). Accordingly, the Ab responses of PM+ women were analyzed by gravidity. Longitudinal analysis showed no difference in Ab levels to FV2 between PM+ primigravidae (PG) and PM+ multigravidae (MG) at both sites (Fig. 2c–d), although PM+ MG had slightly higher Ab levels than PM+ PG at the earliest time-points in Ngali II. Thus, our data suggest that regardless of transmission intensity, primigravid women are at an increased risk of placental malaria at delivery due to the lack of anti-FV2 Ab prior to 7 months of pregnancy. Even though some primigravidae produced Ab levels comparable to PM− MG by 8 months of pregnancy, the Ab response probably occurred too late to clear parasites from the placenta before delivery. Equally interesting, our data also illustrate that some multigravid women living in either the high or low transmission sites remained susceptible to placental malaria because they did not produce high levels of Ab to FV2 early in pregnancy. Thus, Ab levels late in the 1st through the 2nd trimester appear to be a primary determinant of placental malaria status.
To further dissect when during pregnancy strong Ab responses correlated with absence of placental malaria, mean Ab levels in plasma of women in Ngali II were evaluated at two-month intervals (i.e., 3–4, 5–6, and 7–8 months of pregnancy) with respect to placental malaria status at delivery. Women with intermediate to high anti-FV2 Ab levels (MFI >5,000) at 5–6 months and 7–8 months had 2.3 (95% CI, 1.02–4.94; p = 0.05) and 2.0 times (95% CI, 1.01–3.93; p = 0.10), respectively, decreased risk of placental malaria at delivery compared to women with <5,000 MFI anti-FV2 Ab. In summary, higher Ab levels to FV2 correlated with absence of placental malaria.
Ab binding to the surface of placental type IE was evaluated by flow cytometry (Fig. 2e). Antibodies targeting the surface of CSA-adhering IE (7G8 strain) were significantly higher in PM− than PM+ women (p = 0.05) (Fig. 2e). The level of surface binding increased linearly from 4 to 7 months in PM− women; whereas, in PM+ women levels remained low and only increased during the third trimester. Overall, similar results were obtained in assays measuring Ab to FV2 and binding to the surface of IE.
To elucidate if anti-FV2 Ab quality is important in parasite clearance, the proportion of high avidity Ab to FV2 (i.e., Ab remaining bound after treatment with 3M NH4SCN) was determined in plasma from women in Ngali II (Fig. 3) and the women in Yaoundé (n = 25) who were seropositive to FV2. This analysis showed that very few women in Yaoundé developed high avidity Ab, preventing further analysis. By comparison, in Ngali II during the first trimester none of the Ab in primigravidae were high avidity. However, by delivery 17.8±3.9% (mean ± SEM) of anti-FV2 Ab were found to be high avidity (Fig. 3a). Furthermore, during the first trimester, 28.4±5.0% of anti-FV2 Ab in multigravidae were high avidity, and this increased to 40.1±2.1% during the third trimester. As expected, the proportion of high avidity Ab was higher throughout pregnancy in multigravidae compared to primigravidae (p<0.0001; multilevel polynomial regression analysis). Thus, the development of high avidity anti-FV2 Ab is gravidity-related. Importantly, the proportion of high avidity Ab was significantly higher in PM− compared to PM+ women throughout pregnancy (p = 0.0009) (Fig. 3b). That is, the proportion of high avidity Ab in PM− women rose from 34±12% at 3 months to 42±15% at 5 months, and plateaued at 43% until term. In contrast, in PM+ women, only 16±18% of Ab were high avidity at 3 months, 23±16% at 6 months, and 33±12% at the end of pregnancy. Although the proportion of high avidity Ab was generally higher in multigraivd compared to primigravid women, some multigravidae still had placental malaria, suggesting high avidity Ab may be needed early in pregnancy for parasite clearance by delivery. Notably, higher proportion of high avidity anti-FV2 Ab at 5–6 months of pregnancy was significantly associated with absence of placental malaria (p<0.0001). While PM− women still had higher proportion of high avidity Ab at 3–4 and 7–8 months of pregnancy, the differences at these time points were not significant (Fig. 3c). Moreover, women in Ngali II with ≥ 35% high avidity Ab during the 5–6 months of pregnancy had a 7.6 times (95% CI: 1.2–50.0; p = 0.0013) lower risk of placental malaria than women with <35% high avidity Ab. In Yaoundé, the number of women with high avidity Ab was too small for accurate correlation analysis and therefore data are not shown. Thus, at least in the high transmission setting, high avidity Ab to FV2 by the 2nd trimester was found to be important in reducing placental malaria by delivery.
Antibodies that bind to CSA-adhering IE and different DBL domains of VAR2CSA have been associated with improved pregnancy outcomes , –. Nonetheless, in previous studies Ab levels at delivery were not found to differ between PM− and PM+ women , ; in fact, higher levels were often associated with active or chronic placental malaria , . In the current study, Ab levels to FV2 were significantly higher from early (3–4 months) in pregnancy until term in PM− compared to PM+ women (Fig. 2a). Likewise, significantly increased binding of Ab to the surface of IE was observed in PM− women (Fig. 2e). Strong Ab responses to FV2 (i.e., >5,000 MFI) at 5–6 and 7–8 months of pregnancy reduced a woman's risk of having placental malaria 2.3 and 2.0 fold, respectively. Since Ab levels increased during pregnancy (Fig. 1 and Fig. 2), these data help explain the previously reported lack of association between Ab at delivery and absence of placental malaria.
Although the above association was only statistically significant in the high transmission rural village where women were repeatedly infected , the same trend was observed in women living in the low transmission urban city. These results agree with our understanding of infection with VAR2CSA-expressing parasites in that Ab responses to VAR2CSA were low or absent early in pregnancy, increased progressively over the course of pregnancy, and were higher following several pregnancies. Overall, these findings suggest that protection from placental malaria correlates with the acquisition of a mature humoral immune response starting during the first half of pregnancy. Previous results showed that the breadth of the Ab response to the 6 DBL domains increases during pregnancy and a broader repertoire was associated with clearance of placental parasites . The current results show that the magnitude and avidity of anti-VAR2CSA Ab continue to evolve over multiple pregnancies and are positively associated with malaria transmission intensity and clearance of placental parasites. Therefore, the overall “quality” of anti-placental malaria antibodies appears to be strongly influenced by malaria transmission intensity.
This study suggests that primigravidae are most likely highly susceptible to placental malaria as they lack high levels of Ab to VAR2CSA during the 1st and 2nd trimesters. Of interest, 66% of primigravid women in Ngali II failed to develop strong Ab responses to FV2 (FCR3 allele), despite being repeatedly infected during pregnancy. In Ngali II, some multigravid women had placental malaria at delivery and they also had lower Ab levels to FV2. Whether this reflects gaps in the antibody repertoire to achieve adequate Ab breadth  or additional factors that control immune responsiveness to placental isolates such as parasites having multiple var2csa copies  should be investigated.
The comparison of women in the city further highlights that malaria transmission intensity has a profound effect on the maturation of the anti-VAR2CSA antibody response. In Yaoundé, only 33% of primigravidae had Ab responses to FV2 at delivery and only 18% of multigravidae developed strong Ab responses to the antigen (Fig. 1). All of the women were either slide- or PCR-positive prior to 6 months of pregnancy, showing that lack of a response was not due to lack of infection (Table 1). It is possible that Ab to other malarial antigens eliminated IE before the parasite expressed the CSA-binding phenotype and sequestered in the placenta, in which case a humoral immune response to VAR2CSA would not have been induced. The combined results from both transmission settings emphasize that development of a vaccine should not only target primigravid women, but women of all gravidities, especially with the changing epidemiological profile of malaria in Africa.
Taken together, our data demonstrate that women who possessed high levels of Ab to FV2, especially those with high avidity, early in pregnancy are likely to be free of placental malaria at delivery. These antibody responses developed better in women from the higher malaria transmission setting, indicating that malaria transmission intensity influences the evolution of protective anti-VAR2CSA antibodies. While we could only use placental malaria status at delivery as a direct outcome measure of Ab to VAR2CSA, it is important to point out that the process of parasite clearance occurred over time during pregnancy. The sooner a woman eliminates malaria parasites from the intervillous space, the better it is for the pregnancy. Thus, vaccines for VAR2CSA should aim to induce high levels of anti-VAR2CSA Ab spanning the entire course of pregnancy. These results are encouraging as they substantiate the important role of anti-VAR2CSA Ab in the clearance of IE from the placenta.
A prospective cohort study was conducted between 2001 and 2005 in the rural village of Ngali II and in the city of Yaoundé, Cameroon, where malaria transmission is perennial and individuals receive approximately 256 and 13 infectious bites/person/year, respectively , .
Women were recruited at government-operated facilities during the first trimester and written informed consent was obtained. At enrollment, malaria histories were recorded and peripheral blood samples collected. Participants were followed monthly, at which time information on their health and anti-malarial drug usage were recorded and peripheral blood samples were collected. Women who became blood-smear positive for P. falciparum were prescribed anti-malarial drugs and iron-supplement according to the government policy. At delivery, information on delivery outcome and the neonate was recorded, and samples of maternal peripheral blood, placental blood, and a biopsy of placental tissue were collected for parasitological evaluation. Chloroquine was the first line antimalarial when the study began, but due to increasing resistance, artemisinin in combination with amodiaquine were adopted during the last year of sample collection in the 2004. None of the women were prescribed antimalarial drugs within 5 weeks of delivery. The prevalence of HIV seropositivity was ∼9% at the time of the study. The study was completed before the implementation of intermittent preventive treatment and bednets in Cameroon. All participants gave written informed consent. The study was approved by the National Ethics Committee, Cameroon and the Institutional Review Board, Georgetown University. Use of the coded archived samples and data in the current study was exempted from human subject research by the Committee on Human Studies, University of Hawai'i, Mānoa, USA.
Blood smears were prepared using maternal blood collected monthly and at delivery. In addition, smears of blood from the intervillous space and impression smears of the placental biopsy were made. Slides were stained with Diff-Quick and read by two microscopists for parasitemia. PCR was used to identify submicroscopic infections , . A woman was considered to have placental malaria if IE were detected in placental blood or impression smears.
Plasma from all women who met the following criteria were used: information was available on the woman's age, gravidity, and malarial status during pregnancy; confirmed malaria infection detected by microscopy or PCR no later than six months of pregnancy; and ≥4 samples collected throughout pregnancy. In addition, plasma from 20 adult males living at each site was used in the serological assays.
Full-length VAR2CSA was produced in Sf9 insect cells using the baculovirus transfer vector. In brief, the var2csa sequence (FCR3 strain, accession number GU249598) was inserted into the Baculovirus transfer vector pAcGP67-A (BD Biosciences) with a histidine tag on the C-terminal end. Linearized Bakpak6 Baculovirus DNA (BD Biosciences) was co-transfected with pAcGP67-A into Sf9 insect cells for generation of recombinant virus particles. Histidine-tagged recombinant protein was purified on Ni2+-Sepharose columns from the supernatant of Baculovirus infected High-Five insect cells using an ÄKTA-express purification system (GE-Healthcare). Detailed information on expression and characterization of FV2 protein was previously published .
IgG levels to FV2 were measured using the bead-based immunoassay. In brief, saturating amounts (3µg) of recombinant FV2 was covalently coupled to 1 million SeroMAP beads (Luminex Corp.) at pH 7 . The beads were blocked with 1% bovine serum albumin (BSA) in phosphate buffered saline (PBS), pH 7.0 for 24 hr. For Ab measurements, 50µl of plasma diluted to 1∶500 was incubated with 3,000 FV2-coupled beads on a titer plate shaker for 1 hr at room temperature (RT). After washings with 1% BSA-PBS, 100µl of R-phycoerythrin-conjugated affinity purified F(ab')2 goat anti-human IgG (Fcγ specific)(Jackson ImmunoResearch) was added and beads were incubated for 1 hr. After washing, median fluorescence intensities (MFI) for ≥100 beads were obtained using a LiquiChip 100 analyzer (Luminex Corp.).
We used a modified ammonium thiocyanate (NH4SCN) elution protocol ,  and assessed the amount of Ab that remained bound to FV2 after 30 min of incubation with 3M NH4SCN (i.e., high avidity Ab). Our approach aimed to determine the proportion of FV2-specific Ab that were of high avidity, rather than the average affinity of the heterogenous Ab pool in each plasma sample. To minimize the effect of Ab concentration on Ab avidity assessment, the proportion of high avidity anti-FV2 Ab was determined using three plasma dilutions spread over the linear range of detection. Then, the mean proportion of high avidity Ab from all three measurements for each sample was determined. In brief, each plasma sample was diluted to 1∶300, 1∶1,000 and 1∶3,000 with 1% BSA-PBS and 50µl of each dilution was incubated with 3,000 FV2-coupled beads as described above. After washing, bead-Ab complexes were incubated for 30 min with 100μl of PBS or 3M NH4SCN before R-phycoerythrin-conjugated goat-anti-human IgG (Fcγ specific) was added to beads for 1 hr incubation. Percentage of high avidity Ab was calculated by [MFI beads with NH4SCN]/[MFI beads with PBS] x100 and the results from the three dilutions averaged. With this protocol, only a minor variation among the three plasma dilutions was found for the same sample. From assaying over 170 samples, the inter-sample mean standard deviation (SD) is ±4.8% and median SD is ±3.8%.
The protocol of Avril etal. was followed . In brief, CSA-binding IE (7G8 strain) were obtained by panning on CSA . Plasma samples were preadsorbed twice with O+ erythrocytes. Then, 10 million erythrocytes with 5–8% trophozoites were incubated with a 1∶10 dilution of plasma, followed by FITC-conjugated goat (Fab')2 anti-human IgG (1∶100 dilution, PNIM1627, Beckman), evaluated using a LSRII flow cytometer (Becton Dickinson), and data were analyzed using FLOWJO 8.1 software (Tree Star, Inc). Results are expressed as the mean of the adjusted geometric MFI for plasma in duplicate. The adjusted MFI = (IEt-UEt) – (IEc–UEc); where IEt = MFI of IE incubated with test human plasma; UEt = MFI of uninfected erythrocytes incubated with test human plasma; IEc = MFI of IE incubated with USA control plasma; UEc = MFI of uninfected erythrocytes incubated with USA control plasma.
The Student t-test (unpaired) was used to compare age and length of pregnancy between women in the 2 study sites. Fisher's exact test was used to compare the proportion of women who were slide- and PCR-positive during pregnancy, proportion of Ab responders between sites and the significance of relative risk assessment. FV2 Ab levels and high avidity Ab in PM− and PM+ women were analyzed using multilevel polynomial regression analysis. In this analysis, longitudinal FV2 Ab levels and percentage of high avidity Ab were dependent variables, while placental malaria status, month of pregnancy (linear and quadratic), and the interaction between malaria status and time, were independent variables. Results with p values ≤0.05 were considered to be significant.
Competing Interests: The authors have declared that no competing interests exist.
Funding: The work was supported by grants from NIAID, NIH, UO1AI43888 and RO1AI071160 (DWT, RGFL); U19AI089688 (JDS); the intramural research program of the Division of Intramural Research, NIAID, NIH (KS, CL); and Danish National Advanced Technology Foundation and Proof of Concept Foundation DTU (AS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
We thank our participants who made this study possible. We also thank the former and present members of our research team at the Biotechnology Center, University of Yaoundé 1, Cameroon for their excellent work in the field and laboratory.
|1.||Brabin BJ. Year: 1983An analysis of malaria in pregnancy in Africa. Bull.World Health Organ6110051016|
|2.||Brabin BJ,Romagosa C,Abdelgalil S,Menéndez C,Verhoeff FH,et al. Year: 2004The sick placenta-the role of malaria.Placenta2535937815081631|
|3.||McGregor IA. Year: 1984Epidemiology, malaria and pregnancy.Am J Trop Med Hyg335175256383091|
|4.||McGregor IA. Year: 1987Thoughts on malaria in pregnancy with consideration of some factors which influence remedial strategies.Parassitologia291531633334080|
|5.||Guyatt HL,Snow RW. Year: 2004Impact of malaria during pregnancy on low birth weight in sub-Saharan Africa.Clin Microbiol Rev1776076915489346|
|6.||Fried M,Duffy PE. Year: 1996Adherence of Plasmodium falciparum to chondroitin sulfate A in the human placenta.Science272150215048633247|
|7.||Salanti A,Staalsoe T,Lavstsen T,Jensen ATR,Sowa MPK,et al. Year: 2003Selective upregulation of a single distinctly structured var gene in chondroitin sulphate A-adhering Plasmodium falciparum involved in pregnancy-associated malaria.Mol Microbiol4917919112823820|
|8.||Salanti A,Dahlbäck M,Turner L,Nielsen MA,Barfod L,et al. Year: 2004Evidence for the involvement of VAR2CSA in pregnancy-associated malaria.J Exp Med2001197120315520249|
|9.||Muthusamy A,Achur RN,Bhavanandan VP,Fouda GG,Taylor DW,et al. Year: 2004Plasmodium falciparum-infected erythrocytes adhere both in the intervillous space and on the villous surface of human placenta by binding to the low-sulfated chondroitin sulfate proteoglycan receptor.Am J Pathol1642013202515161637|
|10.||Feng G,Aitken E,Yosaatmadja F,Kalilani L,Meshnick SR,et al. Year: 2009Antibodies to variant surface antigens of Plasmodium falciparum-infected erythrocytes are associated with protection from treatment failure and the development of anemia in pregnancy.J Infect Dis20029930619500037|
|11.||Staalsoe T,Shulman CE,Bulmer JN,Kawuondo K,Marsh K,et al. Year: 2004Variant surface antigen-specific IgG and protection against clinical consequences of pregnancy-associated Plasmodium falciparum malaria.Lancet36328328914751701|
|12.||Duffy PE,Fried M. Year: 2003Antibodies that inhibit Plasmodium falciparum adhesion to chondroitin sulfate A are associated with increased birth weight and the gestational age of newborns.Infect Immun716620662314573685|
|13.||O'Neil-Dunne I,Achur RN,Agbor-Enoh ST,Valiyaveettil M,Naik RS,et al. Year: 2001Gravidity-dependent production of antibodies that inhibit binding of Plasmodium falciparum-infected erythrocytes to placental chondroitin sulfate proteoglycan during pregnancy.Infect Immun697487749211705924|
|14.||Fried M,Nosten F,Brockman A,Brabin BJ,Duffy PE. Year: 1998Maternal antibodies block malaria.Nature3958518529804416|
|15.||Gysin J,Pouvelle B,Fievet N,Scherf A,Lépolard C. Year: 1999Ex vivo desequestration of Plasmodium falciparum-infected erythrocytes from human placenta by chondroitin sulfate A. Infect Immun6765966602|
|16.||Khattab A,Reinhardt C,Staalsoe T,Fievet N,Kremsner PG,et al. Year: 2004Analysis of IgG with specificity for variant surface antigens expressed by placental Plasmodium falciparum isolates.Malar J32115242514|
|17.||Barfod L,Dobrilovic T,Magistrado P,Khunrae P,Viwami F,et al. Year: 2010Chondroitin sulfate A-adhering Plasmodium falciparum-infected erythrocytes express functionally important antibody epitopes shared by multiple variants.J Immunol1857553756121078904|
|18.||Jaworowski A,Fernandes LA,Yosaatmadja F,Feng G,Mwapasa V,et al. Year: 2009Relationship between human immunodeficiency virus type 1 coinfection, anemia, and levels and function of antibodies to variant surface antigens in pregnancy-associated malaria.Clin Vaccine Immunol1631231919129467|
|19.||Keen J,Serghides L,Ayi K,Patel SN,Ayisi J,et al. Year: 2007HIV Impairs Opsonic Phagocytic Clearance of Pregnancy-Associated Malaria Parasites.PLoS Med4e18117535103|
|20.||Barfod L,Nielsen MA,Turner L,Dahlbäck M,Jensen ATR,et al. Year: 2006Baculovirus-expressed constructs induce immunoglobulin G that recognizes VAR2CSA on Plasmodium falciparum-infected erythrocytes.Infect Immun744357436016790811|
|21.||Oleinikov AV,Rossnagle E,Francis S,Mutabingwa TK,Fried M,et al. Year: 2007Effects of sex, parity, and sequence variation on seroreactivity to candidate pregnancy malaria vaccine antigens.J Infect Dis19615516417538896|
|22.||Tuikue Ndam NG,Salanti A,Le-Hesran J-Y,Cottrell G,Fievet N,et al. Year: 2006Dynamics of anti-VAR2CSA immunoglobulin G response in a cohort of senegalese pregnant women.J Infect Dis19371372016453268|
|23.||Avril M,Kulasekara BR,Gose SO,Rowe C,Dahlbäck M,et al. Year: 2008Evidence for globally shared, cross-reacting polymorphic epitopes in the pregnancy-associated malaria vaccine candidate VAR2CSA.Infect Immun761791180018250177|
|24.||Guitard J,Cottrell G,Magnouha NM,Salanti A,Li T,et al. Year: 2008Differential evolution of anti-VAR2CSA- IgG3 in primigravidae and multigravidae pregnant women infected by Plasmodium falciparum.Malar J71018190692|
|25.||Resende M,Ditlev SB,Nielsen MA,Bodevin S,Bruun S,et al. Year: 2009Chondroitin sulphate A (CSA)-binding of single recombinant Duffy-binding-like domains is not restricted to Plasmodium falciparum Erythrocyte Membrane Protein 1 expressed by CSA-binding parasites.Int J Parasitol391195120419324047|
|26.||Khunrae P,Dahlbäck M,Nielsen MA,Andersen G,Ditlev SB,et al. Year: 2010Full-length recombinant Plasmodium falciparum VAR2CSA binds specifically to CSPG and induces potent parasite adhesion-blocking antibodies.J Mol Biol39782683420109466|
|27.||Beeson JG,Mann EJ,Elliott SR,Lema VM,Tadesse E,et al. Year: 2004Antibodies to variant surface antigens of Plasmodium falciparum–infected erythrocytes and adhesion inhibitory antibodies are associated with placental malaria and have overlapping and distinct targets.J Infect Dis18954055114745713|
|28.||Fievet N,Le Hesran JY,Cottrell G,Doucoure S,Diouf I,et al. Year: 2006Acquisition of antibodies to variant antigens on the surface of Plasmodium falciparum-infected erythrocytes during pregnancy.Infect Genet Evol645946316621725|
|29.||Khurana S,Verma N,Yewdell JW,Hilbert AK,Castellino F,et al. Year: 2011MF59 adjuvant enhances diversity and affinity of antibody-mediated immune response to pandemic influenza vaccines.Sci Transl Med385ra48|
|30.||Olszewska W,Obeid OE,Steward MW. Year: 2000Protection against measles virus-induced encephalitis by anti-mimotope antibodies: the role of antibody affinity.Virology2729810510873752|
|31.||Schlesinger Y,Granoff DM. Year: 1992Avidity and bactericidal activity of antibody elicited by different Haemophilus influenzae type b conjugate vaccines. The Vaccine Study Group.JAMA267148914941538539|
|32.||Reddy SB,Anders RF,Beeson JG,Färnert A,Kironde F,et al. Year: 2012High Affinity Antibodies to Plasmodium falciparum Merozoite Antigens Are Associated with Protection from Malaria.PLoS ONE7e3224222363818|
|33.||Staalsoe T,Megnekou R,Fievét N,Ricke CH,Zornig HD,et al. Year: 2001Acquisition and decay of antibodies to pregnancy-associated variant antigens on the surface of Plasmodium falciparum-infected erythrocytes that protect against placental parasitemia.J Infect Dis18461862611494167|
|34.||Leke RFG,Bigoga JD,Zhou J,Fouda GG,Leke RJI,et al. Year: 2010Longitudinal studies of Plasmodium falciparum malaria in pregnant women living in a rural Cameroonian village with high perennial transmission.Am J Trop Med Hyg83996100421036826|
|35.||Tutterrow YL,Avril M,Singh K,Long CA,Leke RJ,et al. Year: 2012High Levels of Antibodies to Multiple Domains and Strains of VAR2CSA Correlate with the Absence of Placental Malaria in Cameroonian Women Living in an Area of High Plasmodium falciparum Transmission.Infect Immun801479149022331427|
|36.||Sander AF,Salanti A,Lavstsen T,Nielsen MA,Theander TG,et al. Year: 2011Positive selection of Plasmodium falciparum parasites with multiple var2csa-type PfEMP1 genes during the course of infection in pregnant women.J Infect Dis2031679168521592998|
|37.||Manga L,Robert V,Mess J,Desfontaine M,Carnevale P. Year: 1992Le paludisme urbain a Yaounde, Cameroon: l'etude entomologique dans duex quartiers centraux em Soc R Belg Entomol35155162|
|38.||Snounou G,Viriyakosol S,Jarra W,Thaithong S,Brown KN. Year: 1993Identification of the four human malaria parasite species in field samples by the polymerase chain reaction and detection of a high prevalence of mixed infections.Mol Biochem Parasitol582832928479452|
|39.||Walker-Abbey A,Djokam RRT,Eno A,Leke RFG,Titanji VPK,et al. Year: 2005Malaria in pregnant Cameroonian women: the effect of age and gravidity on submicroscopic and mixed-species infections and multiple parasite genotypes.Am J Trop Med Hyg7222923515772312|
|40.||Fouda GG,Leke RFG,Long C,Druilhe P,Zhou A,et al. Year: 2006Multiplex assay for simultaneous measurement of antibodies to multiple Plasmodium falciparum antigens.Clin Vaccine Immunol131307131317035513|
|41.||Pullen GR,Fitzgerald MG,Hosking CS. Year: 1986Antibody avidity determination by ELISA using thiocyanate elution.J Immunol Methods8683873944471|
|42.||Macdonald RA,Hosking CS,Jones CL. Year: 1988The measurement of relative antibody affinity by ELISA using thiocyanate elution.J Immunol Methods1061911943339255|
|43.||Avril M,Cartwright MM,Hathaway MJ,Hommel M,Elliott SR,et al. Year: 2010Immunization with VAR2CSA-DBL5 recombinant protein elicits broadly cross-reactive antibodies to placental Plasmodium falciparum-infected erythrocytes.Infect Immun782248225620194590|
|44.||Elliott SR,Duffy MF,Byrne TJ,Beeson JG,Mann EJ,et al. Year: 2005Cross-reactive surface epitopes on chondroitin sulfate A-adherent Plasmodium falciparum-infected erythrocytes are associated with transcription of var2csa.Infect Immun732848285615845490|
Table 1 Characteristics of women followed longitudinally
|Ngali II||Yaoundé||p values|
|Number of women||39||50|
|Length of pregnancy (weeks)||39.0±2.5a||39.5±1.7||0.312|
|Proportion of women who were|
|Slide-positive 1 or more timesb||79.5%||54.0%||0.01|
|PCR-positive one or more timesb||100%||100%|
|Percent (%) Slide positivity at|
|3 Months of Pregnancy||36.8%||14.0%||0.087|
|4–6 Months of Pregnancy||36.3%||23.4%||0.299|
|7–9 Months of Pregnancy||34.6%||21.6%||0.197|
a mean ± SD.
b samples were selected from women who were slide and/or PCR-positive at least once during pregnancy confirming that women had become infected.
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