Isabella Quakyi

Bernard Tornyigah
email: bernardtornyigah@noguchi.ug.edu.gh

Pascal Houze

Kwadwo A Kusi

Nathaniel Coleman

Guillaume Escriou

Amos Laar

Michel Cot

Julius Fobil

Gloria Quansah Asare

Philippe Deloron

Abraham K Anang

Gilles Cottrell

Michael F Ofori
email: mofori@noguchi.ug.edu.gh

Nicaise Tuikue Ndam
email: nicaise.ndam@ird.fr

Romain Coppée

Adu Bright

Atikatou Mama

Jérôme Clain

Rachida Tahar

High uptake of Intermittent Preventive Treatment of malaria in pregnancy is associated with improved birth weight among pregnant women in Ghana

Keywords: Plasmodium falciparum, pfdhfr, pfdhps, pfk13, sulfadoxine-pyrimethamine, artemisinin, drug resistance

The pathophysiology of pregnancy-associated malaria is characterized by the sequestration of infected erythrocytes in the placenta mediated by VAR2CSA. Based on several studies, VAR2CSA stands today as the leading vaccine candidate aiming to protect pregnant against the adverse effects of PAM. Conversely, the recent identification of a rare P.falciparum strain expressing a sub-variant of var2csa different from the common variants in circulation and infecting multigravid women living in Benin with high anti-var2csa acquired IgG is raising a question on the effectiveness of a monovalent PAM vaccine. In the absence of a licensed vaccine and in addition to the challenge of low coverage, the world health organization revised its policy and currently recommends pregnant women in moderate and endemic areas to receive a monthly administration of intermittent preventive treatment with sulfadoxine-pyrimethamine (IPTp). This study aims to assess the coverage and impact of the revised IPTp-SP policy on pregnancy and neonatal outcomes, identify some of the challenges facing the IPTp-SP policy and determine antibody functionality against the new P.falciparum variant so to advise on the way forward in PAM vaccine development.

In the first study, we enrolled two different groups of pregnant women in Ghana, attending either the first antenatal consultation or delivery. Using patient's information and liquid chromatography coupled to tandem mass spectrometer to assay for SP uptake, we were able to show that the new IPTp-SP policy is well implemented in our study sites and translates to observed improved birth weight (article 1). However, genotyping SP resistance markers among parasite isolates indicated a high prevalence of mutations at codon 581 (A581G) and 613 (A613S/T) at delivery which resulted in an

x increased prevalence of septuple IRNI-A/FGKGS/T pfdhfr/pfdhps haplotypes among post-SP treatment isolates compared to isolates collected before the initiation of IPTp-SP (article 2).

In the second study, we established and followed a preconception cohort of women seeking to become pregnant for the first time in Benin. This study allowed us to show high prevalence of P. falciparum infection in the first trimester of pregnancy when current prevention tools can not yet be deployed. We identified that most of these infections occurring during the first trimester of pregnancy were derived from persistent asymptomatic and submicroscopic infections prior to conception. This study established the role of persistent sub-patent infections as a major reservoir of PAM in early pregnancy and further justifying the need for new prevention strategies that can cover this period of pregnancy (article 3).

Notwithstanding these challenges, we have, in a third study, deciphered the analysis of IgG subclasses in the STOPPAM cohort that was made up of pregnant women of various pregnancy outcomes. This study allowed us to show that both cytophilic and non-cytophilic IgGs are responsible for the protection against PAM and its adverse effects through different mechanisms. This, therefore, indicates that a future vaccine against PAM should elicit a broad spectrum antibody (article 4).

In the last study, we have undertaken to characterize the functional properties of the new variant of var2csa (T2053) expressed by isolates capable of infecting multigravidae. Results indicate that the parasites with var2csa belonging to this type (T2053) can recognize and bind to CSA, although to low extent compared to those from xi FCR3 and 3D7 variants and that IgG specific to FCR3 and 3D7 var2csa were unable to inhibit their interaction to CSA (article 5).

Conclusion:

The work carried out during this thesis reinforces the fact that although IPTp-SP is well utilized across many African countries, it still faces significant difficulties, particularly on the coverage of the first trimester of pregnancy, where the prevalence of infections is very high. Besides, the development of parasitic mutations associated with more resistance calls into question the effectiveness of the use of SP in the long run. In addition, current efforts to develop a vaccine against placental malaria will likely require a combination of alleles to obtain an effective vaccine on field isolates.

xii Résumé Dans les régions endémiques à transmission perenne, le paludisme à Plasmodium falciparum touche particulièrement les enfants d'âge préscolaire en raison de l'acquisition progressive de l'immunité protectrice. La susceptibilité soudaine des femmes enceintes primigestes aux infections en dépit de cette immunité acquise est une exception importante qui distingue le paludisme associé à la grossesse. Cette complication communément appelée « paludisme placentaire » se caractérise par la séquestration des érythrocytes infectés dans les espaces intervilleux du placenta, entraînant de graves conséquences sur la mère et le foetus. La séquestration des érythrocytes ainsi infectés est médiée par une sous-population de parasites exprimant la protéine VAR2CSA à leur surface, un ligand parasitaire qui assure l'adhésion à la chondroïtine sulfate A (CSA). Cependant, des études ont montré l'acquisition d'une protection au cours des grossesses successives, grâce aux anticorps qui inhibent l'adhésion des érythrocytes infectés à la CSA. Pourtant, récemment, il a été identifié une autre variante de P. falciparum qui infecte les femmes multigestes vivant au Bénin.

En l'absence d'un vaccin homologué, l'Organisation mondiale de la santé (OMS) a recommandé et préconisé, en 2004, la mise en oeuvre d'une stratégie de prévention en trois axes impliquant l'utilisation de moustiquaires imprégnées d'insecticide, le traitement préventif intermittent du paludisme pendant la grossesse (TPIg) avec la sulfadoxine-pyriméthamine (SP) et enfin, le diagnostic et le traitement efficace du paludisme et de l'anémie. Bien que cette politique ait été mise en oeuvre dans plusieurs pays africains, la couverture du TPIg est restée toutefois faible jusqu'en 2012 où elle a xiii été amendée pour une augmentation de doses de traitement, la SP devant être administrée de façon mensuelle à partir du deuxième trimestre de grossesse.

Ce travail de thèse vise à évaluer la couverture et le bénéfice clinique de la nouvelle politique du TPI-SP, à identifier certains des défis auxquels cette nouvelle politique serait confrontée et enfin à déterminer la fonctionnalité des anticorps dirigés contre le candidat vaccin VAR2CSA afin de faciliter le développement du vaccin efficace contre le paludisme placentaire.

Dans la première étude, nous avons recruté dans une approche transversale deux groupes de femmes enceintes au Ghana qui arrivaient soit à leur première consultation prénatale ou pour l'accouchement. En exploitant les informations sur les sujets inclus et en effectuant le dosage des niveaux de SP ainsi que la détection du parasite dans le sang, nous avons été en mesure de montrer que la mise en place du nouveau régime de TPI et son utilisation par les femmes enceintes est effective au Ghana. Les résultats se traduisant par une amélioration du poids de l'enfant à la naissance. Cependant, le génotypage des marqueurs de résistance parasitaire à la SP a indiqué une forte prévalence de l'haplotype quadruple dhfr/dhps et une émergence de mutations aux codons 581 et 613 de la pfdhps. Ces mutations pourraient entrainer rapidement l'apparition de phénotypes parasitaires super résistants à la SP dans nos zones d'étude et par conséquent réduire l'efficacité du TPI.

Dans la deuxième étude, nous avons suivi une cohorte pré-conceptionnelle de femmes au Bénin et observé que la plupart des infections à P. falciparum survenant pendant le premier trimestre de la grossesse, sont issues d'infections asymptomatiques et xiv submicroscopiques présentes chez celles-ci bien avant la conception. Cette étude a établi pour la première fois le rôle du portage asymptomatique des infections persistantes comme un réservoir majeur du paludisme placentaire en début de grossesse.

Une troisième étude portant sur l'analyse des sous classe d'IgG acquises par les femmes enceintes du Bénin contre la région de VAR2CSA présentant un intérêt vaccinal, montre que les sous-classes IgG 3 sont particulièrement corrélés avec la protection contre la survenue de faibles poids de naissance. Les résultats de cette étude suggèrent que le vaccin en développement à base de la protéine VAR2CSA devrait favoriser l'obtention d'IgG cytophile de type 3.

Dans une dernière étude, nous avons entrepris de caractériser les propriétés fonctionnelles des variants majeurs de la région id1-id2 de VAR2CSA, cible vaccinale du candidat vaccin PAMVAC. L'expression des protéines recombinantes de variants id1a-id2a des souches de P.falciparum (3D7, FCR3 et T2053) a été optimisée dans la bactérie E. coli. Les résultats indiquent que le variant (T2053) récemment impliquée dans l'infection des multigestes interagit avec la CSA avec une plus faible affinitée comparativement à ceux des variants FCR3 et 3D7. De

General Introduction

For centuries, malaria represents one of the most significant public health challenges and is still recognized as a significant obstacle to economic development. The disease is caused by protozoans of the genus Plasmodium and of the five species; Plasmodium. falciparum, Plasmodium. vivax, Plasmodium. ovale, Plasmodium.

Malariae, and Plasmodium. knowlesi that infect humans, P. falciparum is the most lethal and prevalent in the World Health Organization (WHO) African region accounting for 99.7% of all malaria cases in 2017; mostly in children under five years and first-time pregnant women (1). The global distribution of these parasites is partly dependent on the distribution of its dominant transmitting vector species (2)(3)(4), environmental conditions (5), and population genetics (6). In human history, malaria is the oldest infectious disease, and over the centuries, several interventions have been coined base on the vector breeding habit, parasite life cycle, and transmission. , the global malaria-eradication program (1955)(1956)(1957)(1958)(1959)(1960)(1961)(1962)(1963)) using dichlorodiphenyltrichloroethane (DDT) (7,8) and later, chemotherapy with chloroquine (9). Although all these interventions were successful and responsible for the eradication of the disease in some areas such as Europe and USA, resurgence of malaria in regions such as India due to ban of DDT (10), the disregard of sub-Sahara Africa in the global malaria eradication campaign and the emergence and spread of chloroquine resistance contributed to the resurgence of malaria in the late 1980s and early 1990s (11,12). In 1998, another attempt to combat the disease was initiated through the Roll Back Malaria (RBM) program with vector control, improved diagnosis, and effective treatment of infected persons as its main foundational pillars (7). Since then, we have observed a significant reduction in malaria-associated morbidity and mortality until the past three years when progress stalled and derailed in some countries. This could be seen in the 2018 WHO malaria report that indicates an increase in malaria among countries with the highest-burden except India which had a reduction of reported malaria cases (1). This increase appears to be intricately linked to instability, poverty and poor health infrastructure in these countries.

These interventions include an initial sanitation program (approximately from

Malaria burden in Ghana

Malaria remains a serious public health challenge in most of sub-Saharan Africa. In the West African sub-region, of which Ghana and Benin are apart, it is estimated that 376 million of the population are at risk of malaria infection with children under-five and primigravid women being the most vulnerable (1).

Ghana has a severe disease burden for malaria with seasonal variations which are more pronounced in the north (13). This implies the entire population is at risk of malaria infection however, transmission is markedly less intense in large urban centers compared to rural areas. Over the decades, several interventions have been employed to reduce the burden of malaria in Ghana, but it still accounted to about 32.5% of all OPD attendances and 48.8% of under-five years admissions in the country (14). Nationwide surveys in 2011 and 2014 also estimated population average parasite prevalence to be 28.0 and 27.0% respectively among children 6-59 months of age (15). Furthermore, malaria attributable mortality among this age group reduced from 14.4% in 2000to 0.6% in 2012 (13). Among this infections, Plasmodium falciparum parasite accounts for over 95% of the infections, while the rest of infections are due to Plasmodium malariae and Plasmodium ovale, which mostly occurs as mixed infections with Plasmodium falciparum (13).

Malaria burden in Benin

Like Ghana, malaria is endemic in Benin, accounting for 40% of outpatient consultations and 25% of all hospital admissions (17). This places severe economic burden on both the family of the infected person and the developmental activity of the country. The World Bank estimates that households in Benin spend approximately one-quarter of their annual income on the prevention and treatment of malaria (16). In Benin malaria transmission is all year round with Anopheles gambiae s.s being the predominant transmission vector, although entomological studies have shown insecticide resistance to carbamates among mosquito vector populations in parts of the country (17,18).

Pregnancy and immune alterations

It is a long-recognized fact that pregnancy increases the risk and severity of infections, despite immunity that may have been acquired prior to it (19)(20)(21). This susceptibility and severity are known to be associated with the immune response shift from cell-mediated responses that contribute to fetal rejection to humoral responses that allow the transfer of antibodies to the fetus. This immune alteration, which is localized at the maternal-fetal interface has been shown not to occur alone but in conjunction with certain hormones such as progesterone and estriol, etc (19,22). Parasitic infections are among those infections whose risk factors and severity are increased during pregnancy. Among these parasitic infections, malaria caused by Plasmodium falciparum, and to a lesser extent Plasmodium vivax has been recognized to be the worst (23,24). Over the past decades, these infections have been associated with a range of adverse maternal and fetal outcomes.

Plasmodium falciparum Pregnancy-associated malaria (PAM)

In sub-Sahara Africa, pregnancy-associated malaria (PAM) is known to be one of the primary causes of low birth weight (LBW) and maternal anemia. The adverse effects associated with PAM is due to the sequestration of infected erythrocytes (IEs) in the placental intervillous spaces (figure 2). This massive accumulation of IEs is mediated by VAR2CSA (25); a parasite ligand that promotes adhesion to placental chondroitin sulfate A (CSA). This physiopathological feature, which led to the definition of placental malaria is particularly frequent in first-time pregnancies in areas of moderate and high P. falciparum malaria transmission areas (26,27) Conversely, successive pregnancies are protected from PAM and its adverse effects through anti-VAR2CSA IgG. However, in low transmission areas, all pregnancies are equally at risk of PAM and its adverse effects (28). Unlike in endemic areas, PAM in low transmission areas is accompanied by symptoms.

Consequences of malaria during pregnancy

The consequences associated with PAM vary with transmission intensity. In moderate to high transmission areas, maternal anemia (29) and low birth weight (30) due to fetal growth restriction and/or pre-term delivery are common effects of PAM.

Besides these, PAM is also associated with abortion, and both maternal and neonatal deaths (31). However, pregnant women gain immunity mediated by IgG with subsequent pregnancies. Contrarily, in low transmission areas, pregnant women do not acquire protection irrespective of the number of pregnancies, and this could result in severe malaria, maternal anemia, pre-term delivery, and fetal loss. Also, the consequences of PAM are exacerbated among HIV infected women (32) who already have their immune system compromised. A review by Ter Kuile and his colleagues indicated a consistent increase in both peripheral and placental malaria (1.58 and 1.66 respectively) among HIV infected pregnant women compared to their HIV-negative counterparts (33). This further increases the adverse effects of PAM on pregnancy outcomes (32). Unlike in HIV-negative women, the adverse effect of PAM is across all gravidae. Prevention of PAM has long been a cornerstone in policy terms, relying initially on weekly chloroquine (CQ) prophylaxis (35). Subsequent to the widespread development of CQ resistance, the Africa regional office of WHO changed the policy to a 3-way system that includes the use of an insecticide-treated net (ITN), intermittent preventive treatment of malaria in pregnancy (IPTp) and effective case management of malaria illness and anemia (36).

Insecticide-treated bednet (ITN)

Insecticide-treated net (ITN) is one of the proven, cost-effective components of malaria prevention through vector control approach. In Africa, ITN has increasingly been used as one of the principal means of preventing vector contact since its first adoption into Gambia's primary national health care system (37), targeting children and pregnant women. However, usage of ITN among pregnant women, especially in most African countries, continues to remain below both the 60% Abuja target and the 80% millennium development goal (MDG) targets. This has been attributed to lack of access, inadequate knowledge, and discomfort (38,39), although some progress is being made towards some of these limitations. One of such is the public or media education on the importance of ITN usage.

Across Africa, the primary source of ITN is through free distribution during antenatal care (ANC) visits, although other sources (voucher schemes, subsidizing the cost or selling at full price) are available and exploited. However, the bulk of evidence suggests that ITNs actually have very little -if any -on PAM (40).

Intermittent preventive treatment (IPTp)

IPTp is a full therapeutic course of an antimalarial drug given to pregnant women at routine antenatal care (ANC) visits, regardless of malaria infection, and is currently the only malaria prevention policy implemented in Africa for the prevention of PAM.

This stems from the assumption that all pregnant women living in stable malaria transmission areas in Africa may be infected (36). To avoid past mistakes that occurred as a result of non-compliance, IPTp is scheduled to be given as a direct observation therapy. Sulfadoxine-pyrimethamine (SP) became the obvious choice for IPTp due to its safety in pregnancy, efficacious, cost-effectiveness, and feasibility as a single dose regimen in both clinical trials and Malawi's national policy (41)(42)(43)(44).

Therefore, in 2004, the WHO Africa office formulated a policy with some clear guidelines, which include IPTp-SP should be administered to all pregnant women at least twice during the second and third trimester (excluding the first trimester), so far as it's given one month apart (36). Since then, several countries in sub-Sahara have adopted and implemented it in different forms (while some countries implemented the administration of 2 doses others administered 3) (45,[START_REF] Fokam | Assessment of the usage and effectiveness of intermittent preventive treatment and insecticide-treated nets on the indicators of malaria among pregnant women attending antenatal care in the Buea Health District, Cameroon[END_REF]. Notwithstanding this discrepancy in policy implementation, IPTp-SP is effective in reducing both peripheral and placental parasitemia, the incidence of LBW as well as maternal anemia across different SP resistance settings (30,[START_REF] Tan | Efficacy of sulphadoxine-pyrimethamine for intermittent preventive treatment of malaria in pregnancy, Mansa, Zambia[END_REF][START_REF] Nwaefuna | Effectiveness of Intermittent Preventive Treatment in Pregnancy with Sulphadoxine-Pyrimethamine against Submicroscopic falciparum Malaria in Central Region, Ghana[END_REF][START_REF] Desai | Impact of Sulfadoxine-Pyrimethamine Resistance on Effectiveness of Intermittent Preventive Therapy for Malaria in Pregnancy at Clearing Infections and Preventing Low Birth Weight[END_REF].

Despite this established beneficial effects of IPTp-SP, uptake of this policy was far from set targets across countries that had adopted the policy [START_REF] Almeida | Field evaluation of the intermittent preventive treatment of malaria during pregnancy (IPTp) in Benin: evolution of the coverage rate since its implementation[END_REF][START_REF] Orish | Prevalence of intermittent preventive treatment with sulphadoxinepyrimethamine (IPTp-SP) use during pregnancy and other associated factors in Sekondi-Takoradi, Ghana[END_REF], with only an estimated 55% of women at risk of malaria receiving at least two doses of SP in 2010 where data were available (52). This observed low uptake was irrespective of the rise in antenatal care visits seen within the same period. Therefore, to harmonize IPTp-SP uptake and ANC coverage, the WHO in 2012 revised the policy to a monthly uptake of IPTp-SP starting as early as possible in the second trimester, so far as it is given one month apart. This policy review also came with some clear guidelines which include; IPTp-SP can be administered even at late stage (after 36 weeks) in the third trimester of gestation without safety concern, even on an empty stomach and it should not be given in conjunction with daily folic acid doses at equal or above 5mg. Among HIV infected pregnant women, cotrimoxazole is currently the primary prophylactic drug administered against opportunistic infections. Thus due to the potential adverse effects, it could cause, the policy is firmly against the administration of IPTp-SP in association with cotrimoxazole [START_REF]WHO | Updated WHO policy recommendation: intermittent preventive treatment of malaria in pregnancy using sulfadoxine-pyrimethamine (IPTp-SP)[END_REF][START_REF] Kapito-Tembo | Marked reduction in prevalence of malaria parasitemia and anemia in HIV-infected pregnant women taking cotrimoxazole with or without sulfadoxinepyrimethamine intermittent preventive therapy during pregnancy in Malawi[END_REF].

However, seven years after this review, IPTp-SP coverage continues to be low among countries that adopted the policy, with 54% of the potential recipient receiving one dose (IPTp1), 42% receiving two doses (IPTp2) and 22% receiving three doses (IPTp3) in 2017 (1). This observed low coverage could be due to the slow adoption and implementation of the updated policy, missed opportunities due to SP stockouts, late or non-attendance of ANC, and lack of knowledge on IPTp by both health providers and pregnant women (45,[START_REF] Hill | Factors Affecting the Delivery, Access, and Use of Interventions to Prevent Malaria in Pregnancy in Sub-Saharan Africa: A Systematic Review and Meta-Analysis[END_REF].

Effect of IPTp on peripheral and placental malaria infection

Placental infection is the most specific indicator of PAM [START_REF] Ismail | Placental pathology in malaria: A histological, immunohistochemical, and quantitative study[END_REF][START_REF] Walker | A model of parity-dependent immunity to placental malaria[END_REF], which is most directly related to all the adverse effects associated with PAM (28,34). Although there could be placenta infections without peripheral infections being detected, several studies have also observed peripheral infections, and until recently, Cottrel and colleagues showed an association between submicroscopic infections and adverse effects of PAM [START_REF] Cottrell | Submicroscopic Plasmodium falciparum Infections Are Associated With Maternal Anemia, Premature Births, and Low Birth Weight[END_REF]. IPTp-SP exerts its impact by clearing existing parasites and also prevents new infections from occurring. The effect of IPTp-SP is more observed among first time pregnant women as they have not developed antibodies that would pevent placenta infections compared to multigravidae women who have developed such antibodies and thus prevent the sequestration of IE in the placenta [START_REF] O'neil-Dunne | Gravidity-dependent production of antibodies that inhibit binding of Plasmodium falciparum-infected erythrocytes to placental chondroitin sulfate proteoglycan during pregnancy[END_REF].

Prior to the general adaptation and use of SP for IPTp in 2004, it was being used for IPTp in some countries in east Africa (41,43), as well as had been used either monotherapy or combination therapy for the treatment of uncomplicated malaria across sub-Sahara Africa in the 1980s and resistance had emerged [START_REF] Basco | Molecular basis of in vivo resistance to sulfadoxine-pyrimethamine in African adult patients infected with Plasmodium falciparum malaria parasites[END_REF][START_REF] Watkins | Treatment of Plasmodium falciparum malaria with pyrimethamine-sulfadoxine: selective pressure for resistance is a function of long elimination half-life[END_REF].

However, while some studies have shown the effectiveness of IPTp-SP on preventing both peripheral and placental infections, others did not observe such effects. In 2009, Harrington and colleagues noted that IPTp-SP failed to prevent both peripheral and placental infections in Muheza, Tanzania, and concluded that IPTp-SP could exacerbate placenta infections in high SP resistant areas [START_REF] Harrington | Competitive facilitation of drug-resistant Plasmodium falciparum malaria parasites in pregnant women who receive preventive treatment[END_REF]. Also, [START_REF] Moussiliou | High rates of parasite recrudescence following intermittent preventive treatment with sulphadoxine-pyrimethamine during pregnancy in Benin[END_REF]. These findings, therefore, suggest that

IPTp-SP has lost some of its parasitological efficacy.

Effect of IPTp on pregnancy outcomes

Anemia is one of the world's most leading physiological hindrances to women from all walks of life. Anemia defined as the reduction in the absolute number of circulating red blood cells (RBCs) is indirectly measured by a reduction in hemoglobin (Hb) concentration, hematocrit (Hct) or erythrocyte count [START_REF] Sifakis | Anemia in pregnancy[END_REF]. WHO classifies anemia into two groups; moderate anemic which occurs when the Hb level is 11<hb≥8g/dl, and severe anemia when Hb level is >8g/dl (65). In stable malaria transmission areas, malaria is a significant contributor to anemia caused by hemolysis of infected and uninfected erythrocytes, although other factors such as helminth infection [START_REF] Larocque | Relationship between intensity of soil-transmitted helminth infections and anemia during pregnancy[END_REF], poor nutrition [START_REF] Unger | Maternal Malaria and Malnutrition (M3) initiative, a pooled birth cohort of 13 pregnancy studies in Africa and the Western Pacific[END_REF] and iron deficiency [START_REF] Allen | Anemia and iron deficiency: effects on pregnancy outcome[END_REF] are also contributing factors. IPTp-SP exerts its effect on anemia indirectly by preventing malaria infections. Though the parasitological efficacy of IPTp-SP is compromised, several studies have observed a positive effect of IPTp-SP on maternal anemia.

Another pregnancy outcome that is affected by IPTp-SP is infant birth weight. In sub-Sahara Africa where PAM is endemic, PAM has been identified as one of the contributing factors in LBW, although other confounding factors such as maternal nutritional status and other diseases have also been identified as playing different roles in infant LBW. In stable malaria transmission areas, PAM increases the risk of LBW, which also increases the risk of neonatal and infant mortality [START_REF] Guyatt | Malaria in pregnancy as an indirect cause of infant mortality in sub-Saharan Africa[END_REF]. In PfPAM, one of the mechanisms associated with LBW is through pre-term delivery mediated by early stimulation of labor by immune responses such as macrophages, cytokines, and antibodies that are elicited due to malaria-parasitized placenta [START_REF] Guyatt | Malaria in pregnancy as an indirect cause of infant mortality in sub-Saharan Africa[END_REF]. Also, intrauterine growth restriction (IUGR) has been shown to cause malaria-associated LBW (30). There have been suggestions that this could directly be due to the prevention of nutrients (glucose) to the fetus as a result of cytotrophoblastic thickening [START_REF] Ismail | Placental pathology in malaria: A histological, immunohistochemical, and quantitative study[END_REF]. Indirectly, IUGR is caused by malaria-associated anemia which results in insufficient oxygen supply to the fetus. IPTp-SP exerts its effect through its clearance of infection that might be present before its uptake and a short-term prophylactic effect of subsequent Pf infections due to the short half-life of SP. Thus, preventing these situations from occurring and thereby allowing the healthy growth of the fetus. This beneficial clinical effect of IPTp-SP continues to be observed across different SP resistant areas [START_REF] Likwela | Sulfadoxinepyrimethamine resistance and intermittent preventive treatment during pregnancy: a retrospective analysis of birth weight data in the Democratic Republic of Congo (DRC)[END_REF].

Effective case management of malaria and anemia

Despite the availability of preventive interventions against PAM, some women may still become infected. Therefore, the WHO highlights the need for prompt diagnosis and effective management of PAM for pregnant women who may become infected (36). This can even be seen in the Abuja declaration of 2000, where African heads of state committed themselves to 60% of pregnant women being treated with effective anti-malarial. Since then, this target has been increased to 80% in 2010 [START_REF]The Abuja Declaration: Ten Years On[END_REF].

Microscopy, which is the golden standard and rapid diagnostic test kits, are the recommended diagnostic tools used in resource-limited areas such as sub-Sahara Africa [START_REF] Vanderjagt | Comparison of the OptiMAL rapid test and microscopy for detection of malaria in pregnant women in Nigeria[END_REF]. Notwithstanding its limitations, these are effective in malaria diagnosis, thus prevent unnecessary administration of drugs. Currently, oral quinine and clindamycin are the recommended drugs for the treatment of uncomplicated malaria during the first trimester, while artemisinin-based combinations (ACT) (i.e.

artesunate-amodiaquine or artemether-lumefantrine) are recommended during the second and third trimester of pregnancy (73) (figure 3). However, in some countries such as Ghana, ACTs became the recommended choice of chemotherapy in the case of quinine failure (74) although it is not yet a WHO recommendation. This could be based on a meta-analysis which found no difference in the risk of fetal loss (miscarriage or stillbirth) or congenital anomalies associated with ACT uptake during the first trimester compared to oral quinine [START_REF] Dellicour | Firsttrimester artemisinin derivatives and quinine treatments and the risk of adverse pregnancy outcomes in Africa and Asia: A meta-analysis of observational studies[END_REF].

Apart from malaria-associated anemia, iron deficiency is another contributing factor to anemia among pregnant women. Although the needed iron could be obtained from dietary products, this source is not efficiently utilized. Thus the WHO's recommendation of prenatal administration of iron and folic acid supplements to prevent iron deficiency anemia [START_REF]WHO | Daily iron and folic acid supplementation during pregnancy[END_REF]. Although folic acid can be obtained from dietary supply, its requirement is increased during pregnancy to prevent neural tube defects [START_REF] Pitkin | Folate and neural tube defects[END_REF]. Thus, the global recommendation of uptake of 0.4mg of folic acid supplement per day [START_REF]WHO | Daily iron and folic acid supplementation during pregnancy[END_REF]. This is well entrenched in the IPTp policy since its inception (36). However, in most African countries, this daily dosage is far exceeded. Although there are no known adverse effects of this excess folic acid supplement uptake on both the mother and fetus, it has been shown to counteract SP efficacy when taken in conjunction by providing an alternate pathway for obtaining folic acid derivatives for parasite replication [START_REF] Hyde | Exploring the folate pathway in Plasmodium falciparum[END_REF]. This invariantly thwarts the effects of SP, thereby increasing the exposure time of parasites to SP which could lead to the emergence and spread of SP resistance. [START_REF] Gesase | High resistance of Plasmodium falciparum to sulphadoxine/pyrimethamine in northern Tanzania and the emergence of dhps resistance mutation at Codon 581[END_REF][START_REF] Naidoo | Mapping 'partially resistant', 'fully resistant', and 'super resistant' malaria[END_REF]. In recent years, additional mutations (mutations at 164, 581 or 613) have also been observed and associated with increased resistance to SP [START_REF] Naidoo | Mapping 'partially resistant', 'fully resistant', and 'super resistant' malaria[END_REF]. Yet the clinical efficacy of IPTp-SP is still observed, thus, the continued use of SP for IPTp as recommended by the WHO. This observed clinical efficacy of SP may be less of a problem than is often assumed, because of the "attenuated vaccine" effect of a partially effective drug. This may allow for natural immunity to be raised. However, several studies are ongoing to find alternative(s) to SP but till now , none is able to show the clinical efficacy of SP in reducing the rate of maternal anemia and increasing birth weights although other drugs such as ACT's have been shown to be effective in clearing parasites [START_REF] Desai | Intermittent screening and treatment or intermittent preventive treatment with dihydroartemisinin-piperaquine versus intermittent preventive treatment with sulfadoxine-pyrimethamine for the control of malaria during pregnancy in western Kenya: an open-label, three-group, randomised controlled superiority trial[END_REF][START_REF] Clerk | A Randomized, Controlled Trial of Intermittent Preventive Treatment with Sulfadoxine-Pyrimethamine, Amodiaquine, or the Combination in Pregnant Women in Ghana[END_REF]. However, the new WHO IPTp-SP policy of monthly uptake of SP could further exacerbate SP resistance and finally the loss of its clinical efficacy. This could be through an increase of drug pressure on already resistant parasites leading to the rise in the overall mutant parasite load.

Infection before conception/pregnancy

Inhabitants living in malaria-endemic areas develop significant naturally acquired immunity to severe malaria during the first five years of life. The breath of this immunity increases with age. Conversely, no immunity is developed in low transmission areas and, thus, severe complications of the disease even at an older age (Figure 4). This partial immunity is mediated by repeated exposure to a repertoire of parasite antigens which is absent or rare in low transmission areas [START_REF] Doolan | Acquired immunity to malaria[END_REF][START_REF] Gatton | Modeling the development of acquired clinical immunity to Plasmodium falciparum malaria[END_REF]. The immunity developed is believed to prevent clinical disease through suppression of parasite load and is what protects the adult populace in endemic areas from the severe form of the disease by conferring protection against highdensity parasitemia [START_REF] Gatton | Modeling the development of acquired clinical immunity to Plasmodium falciparum malaria[END_REF][START_REF] Mendonça Vr De | Immunoregulation in human malaria: the challenge of understanding asymptomatic infection[END_REF]. Regulation of parasite load among inhabitants of endemic areas is due to the adaptive immune system (both B and T-cells) as reviewed by Mendonca and Netta [START_REF] Mendonça Vr De | Immunoregulation in human malaria: the challenge of understanding asymptomatic infection[END_REF].

However, the regulatory mechanisms suppressing parasitemia are partially lost to allow for fetus development after conception. Although the immune system had raised humoral responses to a repertoire of parasite antigens, after conception a subpopulation of parasites express a new variant surface antigen (VAR2CSA) that is not recognized by the immune system thus allowing them to evade and expand. This phenomenon is particular among first-time pregnant women. Until recently, it was assumed that PAM infections were those acquired during the pregnancy. However, a model linking malaria dynamic to observed placenta histology suggested that about 70% of infections carried in early pregnancy are those acquired before pregnancy [START_REF] Walker | A model of parity-dependent immunity to placental malaria[END_REF]. This was based on the assumption that presence of a fully developed placenta increases the risk of placental malaria as it is the stage where any circulating parasites are first able to sequester (in the absence of sequestration-blocking immunity) [START_REF] Walker | A model of parity-dependent immunity to placental malaria[END_REF]. This suggestion was moved further by Berry et

Short term solutions to challenges facing malaria in pregnancy interventions

The current status of SP resistance across sub-Sahara Africa could be traced to its prior use either as monotherapy or combination therapy for the treatment of uncomplicated malaria ( Although all these malaria vaccine candidates are far advanced in its development, they are unable to protect pregnant women against PAM. This is because parasites that infect pregnant women do express different variant surface antigens that have not been accounted in these vaccine candidates. Over the past decade, substantial evidence directs to variant surface antigen 2-chondroitin sulfate A (VAR2CSA) as the principal variant surface antigen (VSA) that mediates placenta infections

Pregnancy-associated malaria vaccine

Several epidemiological studies and basic science had identified, the extracellular region; a highly ordered stretch involving several domains of VAR2CSA to be the primary binding block with CSA and antibodies against it plays a significant role in protective immunity to PAM (118-120). However, the large size and structural complexity of the full-length made it challenging to express it as a possible vaccine.

This challenged further research to identify NTS of the protein (DBL1-3x) as the minimal binding region , and precisely ID1-ID2a segment to be involved in mediating VAR2CSA-CSA binding (119,121). This was through the expression and characterization of small fragments of the VAR2CSA ectodomain. These identify the minimum necessary construct that would be more suitable for vaccine development The first data from the PAMVAC vaccine trial, which was to assess the immunological and safety of this vaccine showed PAMVAC formulated with Alhydrogel or GLA-based adjuvants at 20µg and 50µg dose concentrations were safe, well-tolerated and induced functional antibodies that prevent binding of in vitro VAR2CSA expressing infected erythrocytes from binding CSA in the presence of plasma from vaccinated participants. However, 94 (35.9%) adverse effects were observed in the trial linked to the PAMVAC vaccine (124). Although data from the PAMVAC phase 1a trial looks promising, parasite clonal variability in the field could be a major challenge in a vaccine development aiming for broad protection. Although this challenge had previously been accounted for through the cross-reactivity of the vaccine target, a recent parasite population studies among pregnant women in Benin identified a rare variant of P. falciparum that mainly infected multigravid women who have already gained anti-VAR2CSA antibodies. The fact that these infections were associating with high parasite densities and these parasites express a sub-variant of VAR2CSA that is similar to that of a rare isolate identified for the first time in a study in Asia make it an intriguing observation (125). Molecular epidemiology studies on the N-terminal region of var2csa on isolates from pregnant women in Benin and Malawi subsequently characterized these subtypes of var2csa as particularly distinct (117). In these two African malaria-endemic settings, parasite isolates harboring VAR2CSA variants similar to those of 3D7 have been associated with more severe adverse outcomes, supporting the differential effects of infection with placental parasite strains. The diversity associated with the differential clinical effects suggests that an effective vaccine based on VAR2CSA may require multivalent activity (117)

AIM OF THE STUDY

The aim of this thesis project was twofold and consisted firstly of evaluating current measures to prevent malaria during pregnancy, particularly, the use of sulfadoxinepyrimethamine intermittent preventive treatment in Ghana. In a second step, the project aimed to characterize infections occurring in the first trimester of pregnancy that escapes current prevention measures and study the feasibility of an effective VAR2CSA-based malaria vaccine as a complementary element to prevention strategies.

We studied the implementation in Ghana of the new IPTp-SP policy as recommended by WHO since 2012 and its clinical benefits on pregnancy outcome (Article I). Although these measures made it possible to reduce the prevalence and the densities of malaria infections, it was advisable to study the polymorphism of the parasite genes associated with resistance against antimalarials, particularly in this context of increased pressure by the increase doses of SP and the generalization of the use of ACTs in case management in Ghana (Article II).

The parasite protein VAR2CSA is now incontestably recognized as a serious candidate for vaccine development against placental malaria. The antigenic polymorphism of this relatively conserved protein compared to the other proteins of the PfEMP1 family needs to be taken into account to guarantee the success of the current efforts of its development in vaccine. This approach requires a better characterization of the infections occurring during pregnancy and the indicators of their tropism for the placenta taking into account those occurring during the first trimester of pregnancy (Article III). We have deciphered the antibody response in terms of IgG subclass in a cohort of pregnant women followed in Benin as part of the STOPPAM study and studied their relationship with protection (Article IV). We also addressed the characterization of an antigenically different VAR2CSA-expressing parasite variant as a contribution to optimize the development of an effective placental malaria vaccine (Article V).

ASSESSING THE CURRENT STRATEGIES OF PREVENTING MALARIA DURING PREGNANCY

Objective

Across sub-Sahara Africa, first-time pregnant women are vulnerable to PAM and their adverse effects. As such the WHO recommends a three-way solution to tackle PAM that includes use of insecticide-treated bednets (ITN), intermittent preventive treatment of malaria during pregnancy (IPTp) and effective diagnosis and treatment of malaria and anaemia. However, these interventions are faced with several challenges that include the late arrival of women in antenatal care and, in particular, the first ANC, the low coverage level of intermittent preventive treatment in many African countries, the increasing and spread of parasite resistance to the chemotherapy being used for IPTp, the window of application of this strategy which cannot be put in place before the second trimester of pregnancy. Therefore, several steps (both short term and long term) need to be taken to prevent PAM.

Article I

High uptake of Intermittent Preventive Treatment of malaria in pregnancy (IPTp) is associated with improved birth weight among pregnant women in

Ghana

Objective 1. To assess the degree of coverage of IPTps in two communities in southern Ghana.

2. To determine the beneficial effect of IPTp-SP on pregnancy outcomes.

Key findings

 Early attendance to prenatal clinics by pregnant women in our study sites  High uptake of IPTp-SP with over 60% of pregnant women having taken three or more doses  Uptake of three or more doses of IPTp-SP is associated with an average birth weight increase of 0.165kg  Withholding of IPTp-SP administration to a section of pregnant women based on their G6PD deficiency status

Introduction

Pregnant women continue to be at increased risk of Plasmodium falciparum infection, and thus its harmful effect on both mother and foetus 1 . In sub-Saharan Africa, where the disease is still highly endemic, primigravidae and HIV infected persons represent two of the groups most at risk of infection and the harmful consequences associated with it 2,3 . The World Health Organization (WHO) recommends use of insecticide-treated nets, effective case management, and intermittent preventive treatment of malaria in pregnancy (IPTp) to control and prevent pregnancy-associated malaria (PAM) in sub-Sahara Africa. IPTp was initially a 2-dose regimen of sulfadoxine-pyrimethamine (SP) to HIV-naïve pregnant women 4,5 . Despite the several established advantages of IPTp-SP, even across regions with a wide range of SPresistant parasites 6-11 , uptake of the service by pregnant women was still low compared to ANC visits 6,12,13 . This persistently low coverage in part resulted in a call to action by WHO and its partners, intended to increase coverage to meet the 2010 target of 80% IPTp-SP coverage 14,15 . In addition to this call, other studies revealed a three-course or monthly IPTp-SP administration to be more effective in reducing low birth weight than the original twocourse policy 16,17 . In 2012, the WHO revised the policy to a monthly administration of IPTp 18 . The main goal of this policy change, which was to increase the doses of IPTp during pregnancy, is also dependent on how early women appear at first antenatal care (ANC) visit and the rate of use of these prenatal consultations. Like most malaria-endemic countries in Africa, Ghana adopted and implemented this revised policy, and this has presented an opportunity for a preliminary assessment of the coverage achieved by the new policy and its impact on maternal and neonatal birth outcomes.

Results

Study population characteristics

Overall, 1922 pregnant women aged between 12 and 45 years were recruited in the district hospitals of Maamobi and Kpone on-Sea respectively. This comprises of 987 women at first ANC and 935 women at delivery (Table 1). . Among the women recruited at both first ANC and at delivery, participants' characteristics were similar between the two study sites, except for data on G6PD deficiency at delivery, which was 12.9% (82/635) at Maamobi but the corresponding data were not available at Kpone on-Sea. 2). Thus before first IPTp-SP administration at ANC, 5.9% and 5.0% of pregnant women were submicroscopically infected at Maamobi and Kpone on-Sea respectively.

P. falciparum prevalence in

P. falciparum prevalence in the delivery cohorts

At delivery, parasite prevalence by microscopy was 2.4% (15/635) and 0% at Maamobi and Kpone on-Sea, respectively. Prevalence of infections increased to 4.4% (28/635) at Maamobi and 3% (9/300) at Kpone on-Sea by 18SqPCR, and remained high for ATSqPCR; 12.6% (80/635) at Maamobi and 8.7% (26/300) at Kpone on-Sea. However, placental infection was observed in 0.3% (2/635) of women at Maamobi and 0% of women at Kpone on-Sea by microscopy. Placental infection prevalence rates by 18SqPCR were 2.5% (16/635) at Maamobi and 1.7% (5/300) at Kpone on-Sea, while infection prevalence when tested by ATSqPCR was 6.9% (44/635) at Maamobi and 3.7% (11/300) at Kpone on-Sea (Table 2).

When we compared data from the 18SqPCR and the ATSqPCR, there was 84.6% concordance between the two methods used as all infections detected by the 18SqPCR was also detected by the ATSqPCR except one. However, there was a 15.3% discordance between the two methods mainly due to detection of infection by ATSqPCR among the 18SqPCR negatives (Table 3).

Coverage and impact of IPTp in the delivery cohorts

At delivery, information on IPTp-SP uptake was taken from the participants ANC booklets as a reliable source because of the practice of directly observed treatment. Among the 935 women recruited at delivery, 603 (64.5%) had taken ≥ 3 IPTp-SP doses, 143 (15.3%) had received 2 IPTp-SP doses while 53 (5.7%) had taken 1 IPTp-SP dose. One hundred and thirty-six (14.5%) did not receive any IPTp-SP. When the data were stratified by site, of the 635 participants at Maamobi, 365 (57.5%) had taken ≥ 3 doses, 106 (16.7%) had taken 2

IPTp-SP doses and 39 (6.1%) had taken 1 IPTp-SP dose. One hundred and twenty-five (125) participants did not receive any IPTp-SP, and among them 82 (65.6%) were disqualified at the hospital due to glucose-6-phosphate dehydrogenase (G6PD) deficiency detected by a nitroblue tetrazolium test (Figure 1).

At Kpone on-Sea, 238 of the 300 participants (79.3%) received ≥ 3 IPTp-SP doses, 37 (12.3%) received 2 IPTp-SP doses while 14 (4.7%) received 1 IPTp-SP dose. Eleven (11) participants reported no IPTp-SP uptake due to non-attendance to ANC (Figure 1).

Women who took ≥ 3 IPTp-SP doses had a lower risk of P. falciparum infection detected by qPCR, compared to those who received only 1 or 2 doses, (OR= 0.72, p = 0.36 at Maamobi; OR = 0.69, p = 0.59 at Kpone on-Sea,) (Table 4). In contrast, the overall linear regression analysis showed that uptake of ≥ 3 IPTp-SP doses increase the average birth weight by more than 0.165 Kg. This increase was only statistically significant at Maamobi (P < 0.005) although the same trend was visible at Kpone on-Sea (Table 5). This effect was lower and not significant in women taking only 1 or 2 IPTp-SP doses although an increase in birth weight of about 0.72 Kg was observed as compared with women who did not receive any IPTp-SP treatment.

Sulfadoxine-pyrimethamine concentration measurement

To assess and confirm uptake of SP by pregnant women, we measured SP residual levels among a sub-group of participants from both the ANC and delivery cohorts. For ANC, this was to assess whether pregnant women attending their first ANC at the study sites had taken any SP. In the first ANC group, we measured SP residual levels among 353 and 408 plasma samples from Maamobi and Kpone on-Sea, respectively. Of the samples analysed at Maamobi, sulfadoxine was detected in 99 (28%) of them, with median concentration of 3777 mg/ml and interquartile range (IQR: 263 -38335), while we detected pyrimethamine in 54 women (15.3%) and median concentration of 224 ng/ml (IQR: 18.75 -410.3). At Kpone on-Sea, sulfadoxine and pyrimethamine were detected in 10 (2.5%) plasma samples at median concentration of 2160 mg/ml (IQR: 256.8 -4378) and in 1 (0.2%) plasma sample at a concentration of 216 ng/ml, respectively. Women in whom we detected pyrimethamine were also sulfadoxine-positive, however sulfadoxine was detected in more women. So, we considered the presence of sulfadoxine as a proxy for SP in the analyses.

In the delivery group, we measured SP residual levels in peripheral and also in cord blood to explore whether the amount of SP observed in the mother's peripheral blood could be detected in the cord compartment. SP level in both maternal peripheral and cord plasma was significantly associated with IPTp-SP uptake (Table 6). When the plasma SP levels in the maternal peripheral blood at delivery were categorized into quartiles, the same pattern of improvement in birth weight was observed for women with high concentrations of SP in their blood at delivery. When the data were stratified based on site, this significant association was only observed at Maamobi (Data not shown).

Discussion

Despite the recommendation and implementation of IPTp-SP in some countries in tropical Africa some decades ago, pregnant women are still affected by malaria disease and its effect on their neonates. This is partly due to the low utilisation of the IPTp policy irrespective of high coverage of ANC visits 5,19 . To address this, WHO revised the policy to a monthly administration of SP to reduce inadvertent low or non-uptake of SP 18 . Thus, determining the coverage and effectiveness of this new policy is of utmost importance to its sustainability or otherwise across the different SP-resistant areas in tropical Africa.

In this study, we enrolled two different sets of pregnant women; those who were visiting the health centres for their first antenatal consultation and those coming to deliver at the health centres. The design of this study makes it possible to better capture the characteristics of women appearing at their first ANC and also to determine the use of the services set up for malaria prevention in the delivery cohort. The study design also makes it possible to limit the introduction of potential biases on the envisaged observations. The first observation is the early arrival of women at first ANC, at 18 weeks of pregnancy on average at both study sites, the earliest of which is at 11 weeks. This observation is important and very reassuring for the policy change. Our data which shows over 60% of pregnant women taking ≥ 3 IPTp-SP doses, together with other recent studies in Ghana that report between 87 and 99% IPTp-SP uptake of ≥ 3 IPTp-SP doses 20,21 indicate a high IPTp coverage across Ghana. The observed satisfactory coverage indeed results from the effective campaigns by the Ghana Health Service (GHS) for the IPTp-SP policy. Measuring SP molecules in the blood of women under

IPTp corroborated the documented information on treatment received by the participants.

A high proportion of women (more than 75%) with detectable SP in both peripheral and cord plasma at delivery is also indicative of a high IPTp-SP coverage in Ghana as expected by the new policy. Further, this clearly shows that SP can cross the placenta barrier and raises the question whether these high doses of SP that correlated with that of the mother's compartment are appropriate for the condition of the fetus. However, future studies on the impact of prolonged use of SP in IPTp on the health of the fetus are needed. This is very interesting as this biological measurement correlated with the number of SP doses recorded when the measurement was adjusted for ANC attendance. However, we detected SP in an unexpected and relatively high proportion of women (28%) visiting the urban site for their first ANC, which falls out of line with their declaration. As we did not find this in the periurban site; we cannot exclude the hypothesis of possible unreliable disclosure due to selfmedication or a first undeclared visit in a private clinic. Our possibilities of verifying what these women in this group disclosed before their first ANC attendance at the study health centres are minimal. Thus, the use of this analytical tool could lead to definition of a followup marker for SP uptake.

The significant finding of this study strongly confirms increased birth weight associated with increased SP uptake as recommended by the revised policy. In this case uptake of ≥ . The low prevalence rates of infections observed at delivery compared with those amongst the first ANC group is an indication of IPTp-SPs' curative and prophylactic effect as a whole. However, the fact that taking ≥ 3 doses of IPTp-SP did not show a particular impact on the infection rate at delivery probably suggests that this variable does not provide information on all infections that would have been prevented since initiation of IPTp. This variable can only measure infections observed at delivery which may include more recent infections, while the indirect effect of IPTp that was visible on the improvement of birth weight would better reflect a broader window of coverage.

Sulfadoxine like other sulfonamides is a broad-spectrum antibiotic that is known to likely exert an inhibitory effect against potential non-malarial infections such as sexually transmitted and reproductive tract infections that can cause low birth weight and preterm birth. Thus, SP uptake can sufficiently curb these infections and indirectly improve birth weight as observed elsewhere 22 . This could partially explain why in areas where SP efficacy has reduced, SP continued to improve birth weight outcomes. The effect size is similar in both sites but probably due to lack of power, statistical significance was only achieved at the Maamobi site. A possible explanation may lie in the power of analysis facilitated by a larger sample size of participants that reported of not receiving any IPTp-SP at Maamobi due to G6PD status compared to Kpone on-Sea where virtually every participant received SP, or other possible factors such as maternal nutrition, malaria history or other infections that may be specific to the site but which we are unaware of.

In our study populations the prevalence of microscopically detectable Plasmodium infections was low (less than 4%), however, the use of qPCR revealed the huge difference in the proportion of women who were parasite-positive with microscopy and PCR. The high sensitivity of the 18S qPCR (detection limits of 0.5-3 parasites/µl of blood) and ATS-qPCR (0.06-0.15 parasites/µl of blood) 26 has been an asset for optimal detection of these infections that are missed by conventional microscopy with a detection limit of only 50 parasites/ µl of blood 27 . The reasons for low placental infections compared to peripheral infections are not known, it is possible that all the peripheral infections detected are not necessarily of placental type. Although densities of infection in the placenta are generally higher compared to the peripheral blood, the asymptomatic carriage of certain non-placental type peripheral infections cannot be ruled out. The use of a more sensitive detection PCR in this study, such as ATSqPCR which not only numerically recognizes more targets on the parasite genome than do the 18SqPCR, but also the fact that it was expended over 45 cycles of amplification, allowed to detect these populations probably more prevalent at lower densities.The study demonstrates that low-density parasitemia is very common in high malaria-endemic areas.

To conclude, the new IPTp policy is well implemented and utilized by pregnant women in our study sites confirmed by plasma SP level measurements. Our findings also showed that ≥3 SP administrations of IPTp were associated with increased birth weight as compared to the previous standard 2-dose regimen. Also, as in any observational field study, we did not control for all possible confounding factors. At the same time, this study also reveals that despite the reduction of adverse effects on birth weight by administering of more doses of IPTp-SP, malaria infections remain a threat outside the window of application of IPTp, mainly before women present to antenatal care. Therefore, the need to complement this strategy with an approach that can protect women from infections in the first trimester of pregnancy, such as the use of a vaccine, remains a necessity.

Methods

Study population and design

The study was a cross-sectional hospital-based survey conducted in two distinct communities in southern Ghana; Kpone on-Sea, a semi-urban community and Maamobi, an urban community about 50 Km apart. Malaria transmission in both communities is perennial, with two peaks in April-July and September-November. However, the entomological inoculation rate (EIR) at Kpone on-Sea is 62.1 27 while that of Maamobi is 44.7 28 . At Kpone on-Sea, primary health care is mainly provided by a health centre that also serves surrounding communities. In Maamobi, primary health care service is provided by the Maamobi General Hospital located in the heart of the Maamobi township and also serves several communities in its vicinity. In each site, two independent, HIV-naïve, groups of pregnant women were enrolled in cross-sectional surveys; one survey targeted pregnant women attending their first ANC to get an overview of the appearance of women at first ANC and their characteristics, while the other targeted pregnant women attending the maternity wards for delivery, and was used to study the IPTp coverage and its impact.

Thick and thin blood films were read for Plasmodium species according to standard, qualitycontrolled procedures 29 . DNA extraction was carried out on red blood cell pellets using the QIAamp DNA blood Mini Kit (Qiagen, France) according to the manufacturer's recommendations. P. falciparum parasites were detected and quantified in duplicate by realtime quantitative polymerase chain-reaction (qPCR) targeting the 18S rDNA (18SqPCR) 29,30 and the acidic terminal segment (ATS) of P. falciparum var gene 24 . For 18SqPCR the detection was carried out on 40 amplification cycles. Parasitaemia was further determined by extrapolation of cycle thresholds (Ct) from a standard curve of 3D7 P. falciparum infected erythrocyte culture, and generated simultaneously on each plate. For the detection of parasite DNA with ATSqPCR, amplification was extended over 45 cycles as previously described 24 .

Purified DNA from 3D7 parasite strain was used as a positive control while a negative control with no DNA template was run in all reactions.

Antimalarial drug level measurements

Plasma sulfadoxine and pyrimethamine levels were measured by liquid chromatography coupled to tandem mass spectrometry (TSQ Quantum Ultra, Thermo Fisher, France) with electrospray ionisation in positive mode using an Atlantis T3 (100 mm x 2.1 mm, 3 µm) column (Waters, France). Separation was assessed using a 0.30 ml/min flow rate and a gradient mode using two solvent phases, A (water/methanol/formic acid, 95/4.9/ 0.1% v/v) and B (methanol/formic acid 99.9/0.1% v/v). Specific multiple reaction monitoring (MRM) transitions were selected for detecting sulfadoxine (311.10/245.10) and pyrimethamine (249.10/233.10). Quinide-d3 (328.09/163.04) was used as an internal standard. Separation was achieved in less than 10 min. Using 96-well micro plates (OASE Waters, France), 100 µl of plasma were mixed with 300 µl of acetonitrile containing quinide-d3 (50 ng/ml) as internal standard proteins and phospholipids were eliminated by positive pressure (20 psi during 1 min.). Eluents were evaporated at room temperature, and dry residues were dissolved in 20 mM ammonium formate buffer with formic acid (0.5% v/v) before 10 µl was injected into the system. Method was linear between 10 and 10000 ng/ml for sulfadoxine and between 10 and 1000 ng/ml for pyrimethamine. Lower limit of quantification was 10 ng/ml for both sulfadoxine and pyrimethamine. Upper limits of quantification were 25000 and 5000 ng/ml for sulfadoxine and pyrimethamine, respectively.

For homemade and external controls (WWARN controls), coefficients of variation were below 10%, and bias values were ± 10%. Appropriate dilutions were performed in blank serum for all samples and concentrations higher than 10,000 ng/ml for sulfadoxine or higher than 1,000 ng/ml for pyrimethamine and re-analysed.

Statistical analysis

At delivery, the primary exposure variable of interest was IPTp-SP usage, and it was grouped into 3 categories; women who did not receive any SP, women who received 1 or 2 doses, and women who received ≥ 3 doses of SP. We analyzed the beneficial effect of SP on birth weight based on the number of SP intakes using a multivariate linear regression model.

The risk of P. falciparum infection during pregnancy both at first ANC and at delivery was analyzed using a multivariate logistic regression model. To evaluate the association between SP uptake and residual SP level, we performed a logistic regression after categorizing SP into a binary variable based on the median level. In these analyses, the adjustment factors were gravidity (primigravidae or multigravidae), infection status (positive or negative by PCR), gestational age and SP intake information from the ANC booklets. The Cohen Kappa test was used to test for concordance between the 18SqPCR and the ATSqPCR methods used in P.

falciparum detection. All statistical analyses were done with Stata(version 13.0). All tests were conducted using 0.05 level of significance.

Introduction

In sub-Saharan Africa, Plasmodium falciparum infection represents one of the major causes of low birth weight (1-3). As such, expanded efforts to diminish the burden of the disease on affected populations have been increasingly prioritized over the last two decades (4).

Intermittent preventive treatment of malaria in pregnancy (IPTp) with sulfadoxinepyrimethamine (SP) has been the principal intervention towards that and has been successful across different transmission settings (5-7). However, growing concern over the rise and spread of SP resistance and its impact on the effectiveness of IPTp is rising, especially in eastern and southern Africa, where levels of parasite resistance to SP are high.

Regardless, no alternative has been identified that effects positively on birth weight and is well tolerated (8). As such, the World Health Organization (WHO) updated its IPTp-SP policy from at least 2-doses to a monthly dosage starting from the second trimester ( 9). This increase in SP pressure in pregnant women can potentially favor the selection of parasites carrying mutant haplotypes in the pfdhfr/pfdhps genes that confer increased resistance to SP. This increased resistance is marked by K540E mutation which in conjunction with A581G and A613S/T further confers higher resistance to SP. However, the prevalence of these mutations is lower in west and central Africa compared to eastern and southern Africa. Notwithstanding this, SP-resistant parasites are susceptible to artemisinin (ART)based combination therapy (ACT) which is the first-line treatment in the management of uncomplicated malaria (10) However, since its inception, only few studies have been conducted to determine the spread of resistance to these drugs in Ghana. In this study, we aimed to characterize the genetic diversity of SP and ART resistance markers among parasites from pregnant women in southern Ghana.

Results

Study population characteristics

The general characteristics of pregnant women have been described elsewhere (Quakyi et al, submitted for publication). In total, 223 isolates were evaluated, consisting of 151 isolates from pregnant women enrolled during their first ANC visit and 72 isolates from pregnant women enrolled during delivery (figure 1). Of these, 68 1a). These proportions were similar at both time points (data not shown), except for mutant alleles at codon 581 (A581G) which was preferentially found in post-IPTp-SP treatment isolates (ratio first ANC: delivery of 1:1.7; Table 1b).

Prevalence of pfdhfr and pfdhps haplotypes

Five and nine different haplotypes were detected for pfdhfr and pfdhps genes, respectively.

For pfdhfr, 6.5% (10/153) of isolates carried the wild-type (NCSI) haplotype, while [START_REF] Rodriguez-Morales | Pregnancy outcomes associated with Plasmodium vivax malaria in northeastern Venezuela[END_REF] 2).

Combined pfdhfr-pfdhps haplotypes

Of the successfully sequenced isolates, 126 were analyed for combined pfdhfr and pfdhps haplotypes. Ten distinct haplotypes were observed. Quintuple mutant haplotype, consisting of triple IRNI and double-A/FGKAA mutations, was the most common combined pfdhfr/pfdhps haplotype (63.5%; 80/126), followed by the septuple combined haplotype IRNI + A/FGKGS/T (10.3%; 13/126). The prevalence of other combined haplotypes was low.Only one isolate was wild-type at both pfdhfr and pfdhps loci. The combined quintuple haplotype was observed at similar proportions at both first ANC (63.4%, 52/82) and delivery (63.6%, 28/44). However, there was a high prevalence of septuple haplotype observed at delivery (18.2%) compared to the first ANC (6.1%) (p = 0.03, Chi-squared test; Table 3).

pfk13 propeller-encoding domain

The pfk13 propeller-encoding domain was sequenced for a total of 113 P. falciparum isolates from both time points. Ten isolates failed to generate high-quality sequences and were discarded for further analysis. Among the 103 remaining sequences, ten (9.7%) harbored non-synonymous mutations only found in one (A481C, T535A, Y616S, A621G, L663I) or two (N455D, N458D, I618L, N672D) isolates. Interestingly the non-synonymous mutations N458D and A481C are related to positions at which ART resistance mutations (N458Y and A481V) were previously described in isolates from Southeast-Asia (11).

Another mutation (T535A) was observed at a position found mutated in Southeast Asia (T535M), although its relationship with ART resistance remains uncharacterized. The location of these pfk13 non-synonymous mutations was spotted on the propeller tertiary structure (figure 2). Those were mainly located on the loops connecting blades (Table 4), except the mutation T535A, which was located on the inner strand (strand A) lining the central channel of the domain. None was localized at the shallow pocket, predicted as a possible propeller interaction surface (12).

The Missence 3D tool was used to predict structural alterations caused by non-synonymous mutations. None was predicted to be damaging for the propeller tertiary structure (Table 4).

However, the Dynamut server predicted that five mutations may decrease the propeller domain stability, including mutations N458D and A481C.

Relationships between the number of IPTp doses taken, sulfadoxine-pyrimethamine plasma levels, and prevalence of dhfr/dhps mutations

Based on the declaration and verification on ANC booklet, 9.1% (4/44) of pregnant women reported taking one dose of IPTp-SP, 9.1% (4/44) taking two doses while 63.6% (28/44) of pregnant women took three or more SP doses. Five pregnant women did not receive any SP during their pregnancy.

To explore the relationship between plasma SP levels and the prevalence of drug resistance mutations, we measured SP and ACT residual levels in peripheral plasma at delivery. SP and ACT residual levels were measured in 31 plasma samples with sulfadoxine metabolites being detected in 77.4% (24/31) of samples at a mean concentration of 3467 ± 5543.1 µg/mL, while pyrimethamine metabolites were detected in 16.1% (5/31) of samples at a mean concentration of 4.0 ± 10.2 ng/mL. Women in whom pyrimethamine was detected were also sulfadoxine-positive; however, sulfadoxine was detected in more women. So, we considered the presence of sulfadoxine as a proxy for SP in the analyses.

There was no association between the number of IPTp-SP taken and the frequency of observed mutant alleles or combined haplotypes among women at delivery. When the analysis was carried out with SP level in the blood taken as a quantitative variable between women whose parasites are mutated or not, A581G and A613S/T were associated with increased SP levels (both p = 0.05, Welch t-test). This significant difference was, however, lost when the pfdhfr and pfdhps haplotypes were combined. Finally, we were unable to determine an association between either ACT treatment or level of ACT metabolites and pfk13 mutations, because only one pregnant woman had taken ACT during pregnancy.

Discussion

Gains achieved towards the fight against malaria are now dwindling, especially in sub-Saharan Africa, where most of the burden is felt. This is attributable to the high transmission rate and the low coverage of intervention strategies. In regards to these, the WHO is now recommending an increased administration of SP as part of IPTp to correct the coverage deficit (9,13). Thus, understanding the effect of the increased drug pressure on parasite populations is crucial to the sustainability of the IPTp program, particularly in western Africa, where the prevalence of SP resistance is low (14)(15)(16)(17).

In this study, we characterized parasites obtained from pregnant women coming for their first contact with antenatal care and those of women at delivery who participated in ANCs and have been exposed to several doses of IPTp-SP. Ghana, like other African countries, has adopted and is implementing the new IPTp-SP policy, which advocates an increase in the number of treatment doses, and we have recently reported that this new policy was very well implemented in Ghana (Quakyi et al. submitted for publication). This study is one of the first to assess the SP resistant genetic variation among parasite populations in pregnant women in the context of increased IPTp-SP pressure. One noteworthy observation here is the high prevalence of highly mutated phdhfr/pfdhps haplotypes observed among posttreatment isolates compared to the first ANC. The observation of a clear tendency towards the increase of highly mutated parasites in women still infected at delivery draws attention to a possible selection of these mutant parasites by treatment. Overall, the prevalence of mutant pfdhfr and pfdhps haplotypes conferring SP resistance was high among the parasites isolates in the study participants at both the first ANC visit and at delivery, respectively with 63% and 64% of isolates carrying the quintuple pfdhfr/pfdhps mutant haplotype (IRNIA/FGKAA). This is in agreement with Mouchenhaupt et al. (18) and other studies in Africa (16,17,(19)(20)(21)(22) where the prevalence of the quadruple pfdhfr/pfdhps mutant haplotype (IRNIA/FGKAA) had been reported as very high with almost fixation of the triple pfdhfr mutant haplotype. This predominance of triple phdhfr mutant parasites could be due to the transmission advantage that SP resistant parasites have since acquired in the context of continuous use of the drug (23). When looking specifically at the pfdhps gene, additional critical mutations capable of further impacting resistance to SP, like mutations at codons 581 (A581G) and 613 (A613S/T) of the pfdhps gene have been observed at frequencies close to those reported among children in northern Ghana (24). However, in this study we especially noted that these proportions slightly increased from 9 % and 15 % at first ANC (that is before the initiation of IPTp-SP) to 16 % and 18 % at delivery respectively. The fact that parasites carrying these mutations are more found in women at delivery with higher SP concentrations in their blood suggests that these parasites are better able to withstand these conditions and therefore to withstand SP treatment. When considering these observations, and adding to the fact that parasites carrying the K540E mutation are also present in the study area as recently reported in one isolate from Bioko island in Equatorial Guinea (25), one may wonder whether the conditions necessary for the emergence of full resistance to SP in West Africa will not be gradually favored with the current IPTp-SP strategy. Could the successful implementation of IPTp in Ghana raise fears in this regard, remains a topical issue. These findings warn from the impact of increasing uptake of IPTp-SP on the raising level of resistance among circulating parasites if these few mutants come to disseminate and to become the dominant population. In a meta-analysis study including 42,394 births assessing IPTp-SP effectiveness, mutation at codon 540 (K540E) was associated with high prevalence of low birth weight (26). Therefore, it would certainly be necessary to study the impact on the pregnancy outcome of the pfdhps A581G and A613S/T mutant parasites that seem to be favored in the context of IPTp-SP.

The polymorphism data generated on the pfk13 gene revealed two non-synonymous mutations related to positions known to confer artemisinin resistance in South-East Asia (27)(28)(29)(30). However, the changes in amino acid observed differ from those described in Asia, (34) are suggestive indications of possible ART resistance emergence in Africa. However, several studies have shown that increased parasite survival under ART pressure may also be caused by mutations at other loci such as pfap2-mu, pfcoronin, falcipain 2a and pfubp1 mutations (35)(36)(37).

and
In conclusion, this study shows the high prevalence of quintuple pfdhfr/pfdhps mutations in parasite isolates from two Ghanaian study sites with the development of additional mutations that confer higher resistance to SP. The spread of these new mutations could be facilitated by the continued pressure of IPTp-SP and may impact negatively on its effectiveness. Thus, other interventions to better protect pregnancy from malaria infection and its damaging effects on mother and child should be evaluated now and not wait until the efficacy of SP is completely eroded. On the other hand, the description of mutations on ART resistance-related positions of the pfk13 gene in this study also resonates as an alert to more surveillance on the emergence of resistance against ART derivatives, currently part of the recommended treatment of uncomplicated malaria cases during pregnancy.

Methods

Ethics statement

Ethical clearance was obtained from the Institutional Ethics Review Committee of the

Noguchi Memorial Institute for Medical Research (NMIMR) and the Ethical Review

Committee of the Ghana Health Service. Written informed consent was obtained from each participant.

Study Area and design

The study was a cross-sectional hospital-based survey conducted from December 2015 to May 2017 in two distinct communities in southern Ghana: Kpone on-Sea, a peri-urban community, and Maamobi, an urban community, both within the Greater Accra region in Ghana. Malaria transmission is perennial, with two peaks in April-July, and September-November. A two-parallel cross-sectional enrolment was carried out among pregnant women attending the first antenatal clinic (ANC) and delivery. A detailed description of the study sites and design is reported elsewhere (Quakyi et al, submitted for publication).

PCR assays for the detection and amplification of pfdhfr, pfdhps and pfk13 genes

DNA extraction was carried out on total blood from the peripheral circulation or placenta at delivery using the QIAamp DNA Blood Mini Kit (Qiagen, France) according to the manufacturer's recommendation. The presence of P. falciparum parasites was tested in duplicate by real-time quantitative polymerase chain reaction (qPCR) targeting the 18S rRNA of P. falciparum (38). Subsequently, pfdhfr and pfdhps genes were amplified by nested PCR, and the conditions for amplification were as previously described (39), while that of pfk13 was amplified as previously described by (27). Mutations of the pfdhfr, pfdhps and pfk13 genes in the amplified nested PCR products were purified and detected subsequently by Sanger sequencing (GATC, Cologne, Germany). All sequences generated were analyzed with the Chromas software (Techelysium Pty. Ltd.) and then aligned using MEGA 5.2 (40) and compared with reference genes of the P. falciparum 3D7 genome.

Effect(s) of pfk13 non-synonymous mutations on the structure and stability of the

PfK13 propeller domain

The tools Missence3D (40) and Dynamut (41) were used to predict respectively the structure and stability alterations caused by amino acid changes in the propeller of the PfK13 protein. We used the X-ray crystallographic propeller structure determined at a resolution of 1.5 Ångström (PDB ID: 4YY8, chain A; (42)). Beforehand, all the missing atoms had been added with Swiss PDB Viewer (43).

Antimalarial drug level measurements

Plasma sulfadoxine and pyrimethamine levels were measured by liquid chromatography coupled to tandem mass spectrometry (TSQ Quantum Ultra, Thermo Fisher, France) as previously reported (44). Briefly, using OASE 96-well microplates (Waters, France), 100 µl of plasma were mixed with 300 µL of acetonitrile containing quinide-d3 (50 ng/mL) as an internal standard. Phospholipids were eliminated by positive pressure (20 psi during 1 minute), and eluents were evaporated at room temperature. Dry residues were dissolved in 20 mM ammonium formate buffer with formic acid (0.5% v/v) before 10 µL was injected into the system. This was used to measure metabolites of sulfadoxine, pyrimethamine,

Article III

Objective

 Prove that infections occurring during pregnancy are acquired before pregnancy.

Key findings

 Feasibility of a longitudinal study involving nonpregnant women of child bearing age.

 Infections occurring during early pregnancy (first trimester) are mainly derived from asymptomatic parasite carriage before pregnancy.

starting as early as possible in the second trimester [1]. Consequently, IPTp cannot protect women during the first trimester of pregnancy, although P. falciparum infections are then both frequent and harmful [2].

When a woman exposed to or carrying P. falciparum parasites, even asymptomatically, becomes pregnant, the presence of a placenta provides a novel binding partner for parasite populations that otherwise would be cleared by the spleen. A vaccine dedicated to preventing pregnancy-associated malaria would, therefore, be highly valuable in this context. Such a vaccine to be administered before women become pregnant is currently in the early phase of clinical development [3,4], and

hence not yet available. Identification of infected women during early pregnancy, and a better understanding of the risk factors associated with such early infections may allow their best management by appropriate intervention strategies [5].

In peripheral health centers, malaria diagnosis relies on the rapid diagnostic test or thick blood smear, neither of which is highly sensitive. Previous reports indicated that even infections with very low parasite densities in the peripheral blood, so-called submicroscopic infections, are associated with poor pregnancy outcome with an impact on anemia, prematurity and low birth weight of the offspring in Benin [6], whereas infections detected by means of polymerase chain reaction (PCR) but not with rapid diagnostic tests were not associated with poor outcomes in infants in Malawi, Ghana, or India [7].

In the current study, we enrolled nonpregnant women of childbearing age and followed them up during their subsequent first pregnancy. Using highly sensitive PCR techniques, we show that women infected with malaria while not pregnant, even with submicroscopic infections, were more likely to present with a P. falciparum infection when they become pregnant. Most of these infections during pregnancy resulted from the persistence of the infection acquired before pregnancy.

METHODS

Study Population and Design

The study was conducted in the district of Sô-Ava, 25 km North of Cotonou, Benin. This lagoon area is characterized by a subtropical climate, with 2 rainy seasons (April-July and October-November) and 2 dry seasons. Malaria is hyperendemic, with a mean entomological inoculation rate of 2.05 (standard deviation, 1.3) infective bites per person per 100 nights [8]. Nulligravid nonpregnant women with a desire to become pregnant were enrolled from February 2014 and followed up through conception and pregnancy to delivery. The follow-up ended in February 2016 after the birth of the last newborn. Initially, we aimed to enroll 600 young nulligravid women in a 24-month primary cohort (cohort 1) to identify 50 women who become pregnant and enter a secondary cohort (cohort 2). After enrollment in the primary cohort, women were visited monthly at home to record the first day of last menstrual period, and to perform a urinary pregnancy test (One-Step; International Holding Corp). When the follow-up of the primary cohort ended, nonpregnant women were offered the opportunity to consult a gynecologist for fertility status assessment. All women recruited lived in the Sô-Ava Commune and were of the "Toffin" ethnic group.

At enrollment in the primary cohort, demographic and socioeconomic characteristics were collected. Malaria screening was performed. Once women entered the secondary cohort, they were seen monthly at each scheduled antenatal care visit until delivery and at a final visit 6 months after delivery. Pregnant women were managed according to Benin Ministry of Health guidelines, with supply at the first antenatal care visit of a kit including iron and folic acid, mebendazole for deworming, a longlasting insecticide-treated net, and the first dose of SP for IPTp to take in the second trimester. Although mosquito nets were distributed to all pregnant women, we could not verify their use, but the strong mosquito nuisance on the lagoon might account for high bed net usage. Maternity staff were encouraged to administer ≥3 doses of IPTp with SP (IPTp-SP) from the second trimester onward. At delivery, newborn characteristics were collected. Maternal capillary and placental blood samples were collected. At each visit, 50 µL of blood was transferred to Whatman 3MM filter paper for DNA extraction. Plasmodium falciparum infections were confirmed by microscopic examination of Giemsa-stained thick blood smears, and parasitemia was recorded as the number of asexual parasites per microliter of blood.

Parasitological Determinations by Microscopy and Real-time PCR

Thick and thin blood films were read for Plasmodium species according to standard, quality-controlled procedures [9]. The presence of P. falciparum was also tested in duplicate using realtime quantitative PCR that targeted the 18S ribosomal DNA gene [9,10]. Samples without amplification after 40 cycles of amplification were considered negative. Parasitemia was quantified by extrapolation of cycle thresholds from a standard curve of DNA from 3D7 P. falciparum ring stage-infected erythrocyte culture. A negative control with no DNA template was assayed in all reactions. Plasmodium falciparum infections detected with quantitative PCR, but not with microscopy, were considered submicroscopic [6].

Molecular Genotyping of the Polymorphic Genes msp1 (Block 2) and msp2

Analysis of msp2 (3D7 and FC27 allelic families) and msp1 block 2 (K1, MAD20, and RO33 allelic families) of P. falciparum were performed by nested PCR, as described by Snounou et al [11]. The study was performed on infected samples collected at enrollment in cohort 1 (before pregnancy), and then in the same women at recurrent infections during pregnancy until IPTp intake. Genotypes were compared in each woman, according to band size and number for each of the allelic families of msp1 and msp2. Gel photographs were scored by visual comparison of DNA fragments obtained from baseline and during pregnancy. Unamplified samples and those showing the same msp2 profile on both consecutive samples were analyzed on the msp1 block 2 markers. An infection was considered persistent when identical prepregnancy allele(s) were found in pregnancy samples, the 2 patterns being either completely identical or containing some missing or additional genotypes. Infections were considered new if the allelic pattern at both loci differed completely between the prepregnancy and pregnancy samples.

Statistical Analysis

The prevalence of Plasmodium infection at different time points was determined according to the number of pregnant women seen at that particular time point. We initially used univariate analysis to present the baseline characteristics of the study population. Then we analyzed the risk of P. falciparum infection during pregnancy based on parasitological status at enrollment, using a logistic mixed regression model. The analysis was adjusted for gestational age and parity status. All statistical analyses were done with Stata software, version 13.0 (StataCorp). All tests were conducted using a .05 level of significance.

RESULTS

Baseline Characteristics of Study Population, Sample Collection, and Follow-up

A total of 275 nulligravidae were included in cohort 1. Within a year, 73 women became pregnant, and 68 were included in the secondary cohort; thus, recruitment to the primary cohort was terminated. The time elapsed between inclusion and occurrence of pregnancy was 3 months on average (median, 66 days; interquartile range, 43-130 days). Of the 68 pregnancies included, 15 withdrawals of consent and 16 spontaneous abortions or obstetric pathologies led to 37 deliveries, including 2 neonatal deaths (Figure 1). The monitoring of the primigravid cohort was more complex than anticipated and resulted in high and unexpected attrition. The characteristics of women admitted to cohorts 1 and 2 are presented in Table 1.

P. falciparum Burden in the Study Population

Sixty-eight nonpregnant women who became pregnant for the first time were included for further follow-up (Table 1). The prevalence rates of P. falciparum infection at inclusion in cohort 1 were 15% by microscopy and 66% by real-time PCR targeting the 18S ribosomal RNA gene. At inclusion in cohort Figure 1. Study profile 2, the first month of pregnancy, these rates were 12% and 58%, respectively. The prevalence rates of microscopic infections increased throughout the first trimester of pregnancy, reaching 29% before the introduction of IPTp at about the fourth month of pregnancy. The increase in rates of microscopic infections during this period is clearly in opposition to that of submicroscopic infections, which tended rather to decrease. After IPTp administration, all types of infections decreased, but they increased again toward the end of the pregnancy, especially submicroscopic infections (Figure 2). When cohort 2 is stratified into 2 groups according to parasitological status at enrollment, the follow-up patterns show a lower prevalence of microscopic infections in women who were negative at enrollment, compared with those who had parasites before pregnancy (Figure 3). This observation is confirmed by logistic regression analyses that demonstrate an increased risk of infection in women who were infected before pregnancy. Comparable risks were observed when all microscopic infections were recorded during pregnancy (odds ratio, 6.5 [P = .047] for microscopically positive at enrollment and 3.51 [P = .05] for submicroscopic at enrollment) (Table 2). Similar trends were observed when only infections recorded before IPTp initiation were considered.

Genotyping of Recurrent Infections

From Women Infected at Baseline Consecutive samples from 18 women presenting with a microscopic (n = 7) or submicroscopic (n = 11) infection at both enrollment and at least once before IPTp uptake were sequentially genotyped by amplifying the msp2 and msp1 (block 2) genes. The mean multiplicity of infection (standard deviation) was 4.3 (1.4) before pregnancy, and 3.1 (1.5) during pregnancy. Overall, persistent infections with ≥1 common allele before and during preg-nancy were more frequent than new infections (13 [72%] vs 5 [28%]). Moreover, among the 7 women microscopically infected before pregnancy, infections in all but 1 remained detectable by microscopy, and the mean parasite density increased >20-fold, from 253/µL to 5411/µL. All 11 submicroscopic infections before pregnancy became microscopically detectable during pregnancy, with a mean parasite density of 1228/µL (Table 3).

DISCUSSION

Susceptibility to malaria infections regarding the parasite prevalence and parasite density is increased during pregnancy, representing a major public health problem, with weighty consequences for the mother, the fetus, and the newborn. Understanding the risk factors associated with the occurrence of malaria infections during pregnancy is critical to the development of effective preventive strategies. The WHO recommends that pregnant women use insecticide-treated mosquito nets and IPTp-SP in all areas of Africa with moderate to high malaria transmission. Based on the currently available piece of evidence, the preventive clinical efficacy of IPTp-SP in pregnant women has been demonstrated and is conserved even in areas where P. falciparum-resistant genotypes with quintuple dhpf/dhps mutations are prevalent, but it is vanishing for those with sextuple ones [12], although the parasitological efficacy is compromised [13]. IPTp-SP for pregnant women reduces episodes of maternal malaria, maternal and fetal anemia, placental parasitemia, low birth weight, and neonatal death [14,15]. However, this strategy is also facing the problem of its application safety window, which excludes the first trimester of pregnancy, when infections are equally harmful to a pregnancy.

In the current study, we enrolled nulligravid women with a desire to procreate, and we followed them up until conception and throughout their first pregnancy. This study thus made it possible to characterize the dynamics of P. falciparum infections in this population of first-time pregnant women, those most at risk of malaria infections. The first observation is the demonstrated feasibility of this kind of longitudinal study in Africa, which is among the first such studies. In this study design, a fertility rate of 26.5% (73 of 275 women) within the window of observation was observed, a very satisfactory indicator for future studies to evaluate the effectiveness of preventive strategies deployed during a preconceptional period. Our findings on the dynamics of malaria infections are in line with past notions of increased susceptibility during pregnancy, with a 2-fold increase in the prevalence of microscopic infections between baseline and the first trimester of pregnancy, before IPTp initiation. We also confirmed that submicroscopic infections are more frequent than microscopic infections, especially before pregnancy. During the first trimester of pregnancy the relative prevalence of submicroscopic and microscopic infections evolved differently, so that the overall prevalence of infections remained roughly constant.

Our study clearly shows a previously unknown association between the parasitological status of women before pregnancy and the occurrence of microscopic infections subsequently in a future pregnancy. Furthermore, we show that infections occurring during early pregnancy in areas with high transmissions, such as Benin, are mainly derived from Figure 2. Prevalence rates of Plasmodium falciparum infections at inclusion before pregnancy, each month of pregnancy (M1-M9), and 6 months after delivery (M15), determined using microscopy or real-time polymerase chain reaction targeting the 18S ribosomal DNA gene (for submicroscopic infections). The number of women followed up is indicated above each bar. asymptomatic carriage of parasites before conception, including infections at very low densities, commonly known as "submicroscopic." Although it has been suggested that a large percentage of malaria infections carried in early pregnancy might be acquired before pregnancy [16,17], no formal demonstration has been reported so far. These suggestions were based on seasonal transmission models and did not rule out the total lack of transmission during the dry season. More recent work by our team also suggested that women infected before pregnancy were more likely to be infected during early pregnancy, but it did not rule out the possibility that this was not due mainly to an increased exposure [18].

In our study, parasitological status was determined by microscopy and PCR at the time of inclusion in both cohorts Table 1). The primary cohort was recruited mainly during the dry season for logistical reasons. Inclusion in the secondary cohort occurred at the time of onset of pregnancy, mostly during the rainy season, when malaria transmission was the highest. Although we could have expected a higher prevalence of microscopic and/or submicroscopic malaria infection among young women included during the rainy season, malaria prevalence was similar in the 2 cohorts before increasing during the first trimester of pregnancy. However, the link established in the present study between these parasite isolates and those present before pregnancy by molecular genotyping definitely comes to demonstrate and establish this fact (Table 3), clearly pointing to asymptomatic carriage before pregnancy as a major factor of infection during early pregnancy, certainly more substantial than transmission variations. Although the WHO is currently recommending the universal use of bed nets to prevent malaria across sub-Saharan Africa, the usage has consistently been lowest in adolescents and young adults [19,20].

Table 1. Baseline Characteristics of Women admitted to Cohorts

The current study identifies women of childbearing age as a target of choice for deploying specific strategies to reduce infections during pregnancy. Epidemiological evidence of a high prevalence of asymptomatic infections (then untreated) in adolescents and young women, before first pregnancy, further support this observation [19,21]. Thus, a preventive strategy could involve the use of drugs in the short term or the use of long-term effective vaccines, when available, in the long term. A vaccine earmarked for preventing malaria during pregnancy, to be administered to adolescent girls, would probably be an intervention of choice, but this is not currently available although it is in the early phases of clinical testing [3,4]. Alternatively, presumptive administration of a curative antimalarial regimen, probably using an artemisinin-based combination therapy, could be effective in clearing parasitemia in the preconceptional period, and consequently in reducing the burden of malaria parasite infections during the first trimester of pregnancy, before initiation of IPTp. The exact target population and time frame of this treatment administration remain to be determined.

pregnant specifically express the var2csa member, in contrast to parasites infecting nonpregnant individuals [23,24]. Our observations strongly suggest that these pregnancy-specific parasite phenotypes, most probably representing a minor population in the parasite before pregnancy, can be rapidly selected for in vivo when favorable physiological conditions are met. In this case, the appearance of the placenta will allow the exposure of such parasites to chondroitin sulfate A receptors that are specific to this organ. Such phenotype switching is widely reported with in vitro manipulations, with the panning and generation of selected cytoadherence phenotypes from the same parasite clone [25,26]; no experimental data from in vivo conditions is currently available. Although our current findings do not properly demonstrate this phenotypic switch in vivo, the persistence of preconceptional genotypes and their expansion during pregnancy strongly suggest it.

An additional limitation involves the limited sample size of pregnant women who were followed up. However, the study brings a proof of concept that such a preconceptional cohort is achievable in an African context. Importantly, this allows the study of the very first weeks of pregnancy, a time frame that has hitherto never been investigated. With this sample size, we were still able to demonstrate that malaria infections before pregnancy persisted during pregnancy and were potentially harmful.

Table 2.

Risk of Plasmodium falciparum Infection During Pregnancy, by Parasitological Status at Enrollment, Determined Using a Logistic Mixed Regression Model

Clonal phenotypic variation in P. falciparum, supported by the mutually exclusive expression of genes from multigene families, such as var genes, is known to be the mechanism underlying the variable clinical forms of P. falciparum malaria [22]. Several studies have showed that parasites infecting Abbreviations: CI, confidence interval; OR, odds ratio. b Parasite density as defined by microscopy.

To conclude, we found a substantial burden of microscopic and submicroscopic malaria infection in nulligravid women from south Benin. When these women became pregnant, and before first IPTp administration, the overall prevalence of infections remained roughly similar, but the proportion of microscopic infections, with a higher parasite density in blood, clearly increased. Infections occurring during early pregnancy were derived mainly from parasites infecting a woman before conception. Preventive strategies targeting nonpregnant women with a desire for conception need to be designed to allow the best management of this population before contact with antenatal clinics. The number of genotypes was derived from the analysis of msp1 and msp2 allelic families after genotyping (see Methods). Persistent infections were defined as prepregnancy alleles (either all or not) present in pregnancy samples. Reinfections where defined as fully different allelic patterns between the prepregnancy and pregnancy samples.

Notes

Introduction

Plasmodium falciparum malaria continues to be a significant human disease burden throughout the tropics. In sub-Saharan Africa, placental malaria (PM) is known to cause low birth weight and maternal anemia (1,2). The adverse effects associated with PM are due to the sequestration of infected erythrocytes (IEs) in the placental intervillous spaces. The often massive accumulation of IEs is mediated by VAR2CSA, a parasite ligand that promotes adhesion of IEs to placental chondroitin sulfate A (CSA) (3)(4)(5). This pathophysiological feature, which led to the recognition of placental malaria as a specific syndrome, is particularly frequent in first pregnancies in areas where P. falciparum malaria is highly endemic (6). Women who have had PM develop anti-VAR2CSA IgG antibodies that protect them from the adverse effects of PM during subsequent pregnancies, making this protein an attractive target for vaccine development (4,7). Anti-VAR2CSA IgGs have been shown to inhibit the binding of VAR2CSA-expressing infected erythrocytes to CSA (8)(9)(10). Further studies identified the Id1-DBL2X-Id2 region, in the N-terminal part of VAR2CSA, as being the minimal binding domain mediating binding between VAR2CSA and placental CSA (9), thus making it appropriate for the development of a VAR2CSA-based vaccine with an antigenic construct easier to produce in recombinant form. A Phase 1 clinical trial has recently been completed with such an antigenic construct (10).

Our team has already demonstrated in a longitudinal study that high levels of IgG with specificity for the N-terminal part of VAR2CSA present in women early in pregnancy were associated with better pregnancy outcomes (11). However, information is lacking on which of the IgG subclasses targeting the vaccine construct are responsible for protective immunity, and on the dynamics of such protective antibody levels during pregnancy. Here, we used samples from a group of women whose pregnancy outcomes were well characterized in the STOPPAM study in Benin and from whom consecutive blood samples and clinical data were available from the time of inclusion until delivery. Using measurements of naturally acquired antibody targeting the PAMVAC vaccine Id1-Id2a recombinant antigen on consecutive plasma samples, we analyzed the link between IgG subclasses with different pathological outcomes of pregnancy.

Methods

Study site and population

The current study made use of samples collected from a subgroup of women drawn from the STOPPAM project initially designed to characterize the pathology of placental malaria, to determine the significant determinants for optimal use of IPTp and for vaccine development against pregnancy-associated malaria. A detailed description of the STOPPAM study design has been reported (12). For the current work, 470 women were included based on their pregnancy outcome as previously reported (13). This sub-group included women who had placental infection at delivery, were anemic, whose infants had low birth weight, in whom intrauterine growth retardation was identified based on fetal growth curves derived from ultrasounds to define children born small-for-gestational-age (SGA), or whose infants were born prematurely. Another group of pregnant women from the same area with no pathological outcome at delivery was also included. Briefly, babies were classified as premature if their gestational age, assessed by ultrasound before 24 weeks of gestation, was <37 weeks at delivery. Fetal growth restriction was approximated by low birth weight for gestational age. A baby was defined as SGA if the birth weight was below the 10 th percentile of fetal weight for the gestational age (14). Maternal anemia at delivery was defined as a hemoglobin level below 10 g/dl. Placental malaria was defined as a positive placental blood smear (15). At inclusion and each antenatal or emergency visit, a thick blood smear was processed for malaria diagnosis. Venous blood was collected, and plasma samples were separated and frozen at -80°C for further use. All women diagnosed with P. falciparum infection by thick blood smear during the follow up were treated with quinine according to national guidelines applicable at the time of the study.

Ethical statement

The STOPPAM study obtained ethical approval from two independent institutional

Total IgG and subclasses quantification

To measure antibody response against VAR2CSA, we used the PAMVAC vaccine candidate; a recombinant protein representing the ID1-ID2a construct of VAR2CSA from the FCR3 variant of P. falciparum (10,16). Antibody levels with specificity for the ID1-ID2a recombinant protein were determined by a previously described indirect ELISA protocol (7,10). In brief, antigen-coated (0.5 µg/mL) and bovine serum albumin

Results

The STOPPAM cohort subgroup used in this work are Beninese pregnant women (mean age, 26 years) followed up during the course of pregnancy. Among the 470 women included in this study, 11 (2.3%) attended 3 ANV while 415 (88.3%) attended all the 5 scheduled visits (inclusion, ANV1, ANV2, ANV3, and delivery). Amongst the study cohort 78 (16.6%) were primigravidae, 63 (13.4%) had evidence of placental malaria at delivery, and 82 (17.4%) were anemic at delivery. Forty-nine (10.1%) women gave birth to infants with LBW (<2500g), 53 (11.3%) with SGA, 24 (5.1%) women gave birth prematurely (before 37 gestational weeks), and 272 women had no pathological outcomes. The characteristics of women who had their IgG profiles determined are presented in Table 1.

Antibody profiling and factors associated

Distribution of IgG subclasses show that a cytophilic IgG1 and IgG3 were measured in a high number of women compared to IgG2 and IgG4 (Figure 1a). Analysis of the seroprevalence of the different IgG subclasses shows that IgG1 and IgG3 were the most prevalent with rates ranging from 49.6% to 53.8% for IgG1 and from 27.6% to 27.4% for IgG3 according to the time of pregnancy. IgG2 and IgG4 were less prevalent with rates ranging from 8.3% to 12.1% and from 3.4% to 8.9%, respectively (figure 1b).The ranges were larger for subclass measurements compared to total IgG leading to larger dispersion of values (Table 2). When taking into account repeated measurements made on consecutive samples of the same woman in the analyses, plasma levels of IgG (both total and subclasses) gradually decreased over the course of pregnancy except IgG4 which was consistently low throughout. (Table 3).

When comparing levels by gravidity, it was clear that total IgG levels remained consistently higher in multigravid women compared to primigravidae, in both univariate and multivariate analysis. However, this observation was not so clear for individual IgG subclasses. When taking into account infections that were documented during the follow-up in relation to the time of antibody measurement, a high level of all subclasses were consistently observed in infected women, even though this reached significance only for IgG1 and IgG3. A similar observation was made for antibody subclass levels and parasite density of the infections documented during follow-up, but statistical significance in multivariate analysis was reached only for total IgG (Table 3) (P = 0.028) and IgG2 (P = 0.03).

Relationship with pregnancy outcomes

To compare the level of IgG during pregnancy in women with well-defined pregnancy outcomes, we performed a hierarchical mixed model analysis and factors with pvalue < 0.2 were further incorporated into the final multivariate model (Table 4). For total IgG, low levels of antibodies were consistently observed throughout the followup in women who delivered babies with low birth weight in univariate analysis (P = 0.014). This observation was further confirmed in the multivariate model (P = 0.004).

However, for the other adverse pregnancy outcomes, no significant difference in the antibody levels of total IgG with specificity for the ID1-ID2a PAMVAC antigen was observed. We then tested in a similar way the antibody levels of the IgG subclasses. For IgG1 and IgG2, no significant differences were observed with the different pathological markers at delivery. Levels of IgG3 antibodies were consistently low in women who delivered low birth weight babies compared to those who delivered babies with normal birth weight (multivariate analysis, P = 0.01). Throughout pregnancy, IgG4 antibody levels were low in women presenting with placental infection at delivery compared to women without parasites in their placenta (multivariate analysis, P = 0.03). However, no significant difference in IgG3 and IgG4 antibody levels was observed in the other groups with poor pregnancy outcomes.

Discussion

It is widely documented and accepted that plasma antibodies targeting infected erythrocytes expressing VAR2CSA protect against PM in a parity-dependent manner (18,19,11,20). The difficulty of expressing the recombinant protein representing the full length of the VAR2CSA protein led researchers to characterize its binding properties with CSA and thus to identify the minimum necessary construct that would be more suitable for vaccine development. This allowed the development of the PAMVAC vaccine candidate (10) and characterization of antibody responses to the minimal binding pocket mediating VAR2CSA binding to CSA. Just as it is necessary to study the antibody response induced by this particular region of VAR2CSA, our understanding of the acquisition of specific IgG subclasses in women exposed to placental malaria and the role played in mediating protection to poor pregnancy outcomes is equally important.

In this study of a cohort of pregnant Beninese, we determined the levels of total IgG and of IgG subclasses with specificity for the PAMVAC vaccine antigen. We focused on the PAMVAC antigen currently undergoing vaccine development to decipher the specific antibody response that women develop when exposed to placental-type parasites. The levels of total IgG with specificity for the PAMVAC antigen were consistently lower in women giving birth to low birth weight babies, unlike other women, regardless of the gestational age at which the plasma sample was taken.

These results confirmed our initial observations that low levels of total IgG directed to recombinant DBL1X-Id1-DBL2X of VAR2CSA in early pregnancy were associated with an increased risk of low birth weight (11). The current study emphasizes the fact that this particular association with risk of low birth weight persists throughout pregnancy. Moreover, it should be noted that, as was expected, there were relationships between the antibody levels measured here and the level of exposure to malarial infections or the gravidity status of the women, further supporting the relevance of the observations. One noteworthy observation is the association of low cytophilic IgG3 antibody levels with low birth weight, although IgG1 was the predominant antibody subclass. This finding is similar to that of a study in Senegal (21) and suggests cytophilic IgG3 is the main effector in the prevention of LBW, reflecting the observation with total IgG, through either its direct inhibition of infected erythrocyte adhesion to CSA in the placenta or its ability to mediate opsono-phagocytosis by effector cells, or sensitization and activation of natural killer cells and the complement system (19)(20)(21).

As LBW is among the most frequent detrimental outcomes of PM, this is an indication that any vaccine for PM should be designed to elicit IgG3. Interestingly, we also observed that higher levels of IgG4 were associated with a reduced risk of placental infection. The IgG4 subclass unlike IgG3 does not activate complement or act as an opsonin, suggesting that their role in the protection mechanism can only be achieved through the inhibition of infected erythrocyte adhesion to the placental receptor. In helminthic infections, antibody isotype patterns are an indicator of the host's regulatory status, pointing the relative level of IgG4 as the corollary of chronic parasite carriage favored by regulatory cytokines. Pathology of chronic helminth infections are characterized by elevated IL-10 and TGFβ that mediate IgG4 production by B cells (22)(23)(24). In the case of PM, a previous study by Chene et al (13), in the same cohort measured a high IL-10 cytokine among placenta infected women compared to those not infected. Based on the PCR data we previously generated on some samples from this cohort (25), we observed that women with elevated IgG4 levels were consistently subject to low-density (submicroscopic) parasite carriage at 4 different time points during pregnancy, suggestive of persisting chronic infections. As we have always found, in all our studies, that IL-10 is elevated in the presence of P. falciparum infection, whether peripheral (( 26)) or placental (13), this condition may have favored IgG4 production. It is also known that the IgG4 titers fall rapidly following a curative drug treatment as compared to other subclasses (27). In the context of intermittent preventive treatment administered to pregnant women it is possible that this has contributed to disrupting the association with active infections detected by microscopy in this study. However, the plasma levels of IgG1 and IgG2 subclasses appeared here to be more like such markers of infection. The observations from this study further underline the complex mechanism that may result from the antibody responses, whose properties can both mark the presence of an infection and the success of its control. While this study clearly highlights the importance of IgG3 subclass in the acquired protection against placental malaria, its particularly demonstrates for the first time the role that IgG4 could play in this protective mechanism.

In conclusion, the association of IgG3 subclass antibody levels with the risk of low birth weight and that of IgG4 to that of placental infections at delivery, strongly suggest the the mechanism of protection mediated by the antibody response against VAR2CSA uses different IgG subclasses. This observation further suggests that prospective PM vaccine should target the elicitation of both IgG3 and IgG4 to achieve the expected optimal protection. .

Antibody seroprevalence polymorphisms expressed by field isolates constitute a significant challenge to overcome in the design of an effective vaccine. Conversely, our group had recently identified a rare variant of P.falciparum that mainly infects multigravid women living in Benin with high anti-var2csa acquired IgG (11). The fact that these women who have already gained antibodies against var2csa during successive pregnancies become

I n c A N V 1 A N V 2 A N V 3 D e l I n c A N V 1 A N V 2 A N V 3 D e l I n c A N V 1 A N V 2 A N V 3 D e l I n c A N V 1 A N V 2 A N V 3
In order to verify the expression and integrity of ID1-ID2a variants expressed, we performed an SDS-PAGE stained with coomassie blue (Figure 1A). After His-Trap purification, based on the chromatogram we identified fractions that we believed to contain our protein of interest (Figure 1B). These fractions were pooled and gel filtered to obtain pure concentrated proteins (Figure 1C). ID1-ID2a recombinant variants (1mg) and 12mg were incubated in 1x PBS at 4ºC for 48 hours. No protein degradation was observed between the 1mg and 12mg concentrations after SDS-page analysis. Taken together, these results suggest successfully expressed ID1-ID2a variants of VAR2CSA from FCR3, 3D7, and T2053 parasite strains.

Binding capacity of proteins

To assess the functionality of the ID1-ID2a variants expressed, we performed an indirect ELISA to assess binding capacity to CSA using decorin. There was a significant reduction in the binding ability between the ID1-ID2a variant of T2053 compared to similar ID1-ID2a constructs of FCR3 and 3D7 variants (Figure 2).

Reactivity of T2053 ID1-ID2a mice anti-sera to ID1-ID2a variants of FCR3 and 3D7

Mice polyclonal antibodies induced by T2053 ID1-ID2a variant showed high reactivity against its homologous recombinant protein (Figure 3A). However, the reactivity of mice T2053 antisera against the other ID1-ID2a variants of FCR3 and 3D7 parasite lines was low when assessed by ELISA (Figure 3B). Further analysis for the recognition of native VAR2CSA of panned-selected FCR3 and 3D7 laboratory parasite lines showed similar low reactivity of mice T2053-antisera compared to the control (Figure 3C andD).

Cross-reactivity of T2053 ID1-ID2a mice antisera to FCR3 or 3D7 alleles

antibody profiling against the ID1-ID2a variant of FCR3, and that of T2053 showed parity dependent recognition patterns although higher reactivity was observed to ID1ID2a variant of FCR3 compared to T2053 among both gravidaes. This confirms the dominance of FCR3 variants compared to parasites of T2053 types among the study area. Considering the previously reported observations strongly suggesting that these T2053-like variants may emerge in a context of already acquired immunity against common variants such as those of the FCR3 type, it seems plausible that the sites of interaction with the CSA are presented with structurally distorted way with FCR3 and 3D7 variants. This conformational variability may be sufficient for T2053-like variants to bypass the anti-adhesion properties of antibodies targeting the critical epitopes involved in inhibiting CSA binding of FCR3 or 3D7-like strains. Antigenic variation in the ID1 region, which discriminates these variants, therefore might play a key role in this escape mechanism.

The ID1-ID2a variant of FCR3 (PAMVAC) is one of the two promising vaccine candidates against placental malaria which recently completed a phase 1a clinical trial (16). However, the use of this mono-allele vaccine, although having some crossreactivity could mean that genetically divergent parasite from the vaccine strain such as T2053-like variants could escape its effect (20). This, therefore, raises a serious question of achieving effective monovalent VAR2CSA-based vaccine in an era where there is continuous migration of parasites. This preferential infection of multigravid women in the presence of acquired immunity could be due to the ability of the naturally acquired antibodies filtering out parasites that it recognizes, leaving divergent parasites in pregnant women to grow unimpeded. The low affinity of the T2053 variant for the CSA compared to the FCR3 and 3D7 could explain a disadvantage in their fitness, which would make these variants less competitive in primigravid women as compared to those having a better affinity to the placental CSA. As the parasites with higher affinity to CSA would be confronted with the immunity acquired during successive pregnancies, the rare variants would then find favorable conditions to establish in the absence of immunity which is specific to them. Although this study does not properly demonstrate the recognition of T2053 induced sera to its corresponding parasites (data to come), the low reactivity to FCR3 and 3D7 parasite line and competitive ELISA results already clearly show major antigenic divergence.

An additional limitation involves inducing antibodies against the other variants. This limitation is being addressed, but data on it is not readily available to include in this draft.

In conclusion, we show a differential binding pattern as well as a low recognition between ID1-ID2a variants of T2053 and that of FCR3 and 3D7. This suggests that a successful VAR2CSA-based vaccine will likely require a combination of alleles to obtain an effective vaccine on field isolates. Therefore because of their adhesive property they can interact with a multitude of receptors in the host (116). Currently, VAR2CSA, a member of the PfEMP1 proteins, is identified as the undisputed ligand the mediate sequestration of infected erythrocytes in the intervillous spaces of the placenta (27,(118)(119)(120). This sequestration of infected erythrocytes results in maternal anemia and low birth weight (29,30). In the absence of any licensed PAM vaccine, presumptive use of sulfadoxine-pyrimethamine (SP) in intermittent preventive treatment (IPT) has been the preferred choice of preventing the adverse effect of PAM over the past two decades (36,[START_REF]WHO | Updated WHO policy recommendation: intermittent preventive treatment of malaria in pregnancy using sulfadoxine-pyrimethamine (IPTp-SP)[END_REF]. The successful implementation and utilization of this policy would have resulted in reducing the adverse effects of pregnancy-associated malaria on pregnancy outcomes in sub-Sahara Africa.

However, the uptake of this intervention has fallen short of the expected uptake, although utilization of reproductive health has increased during the same period. This low coverage has been attributed to several reasons including late turnout for antenatal care, SP shortages, instability, poor health infrastructure and implementation (126,127).

Also, a meta-analysis indicates a dose-dependent relationship between IPTp-SP and birth weight (128). To harmonize IPTPp-SP uptake and utilization of reproductive health as well as achieve the desired effect of IPTp-SP, the world health organization (WHO) in 2012 revised the IPTp policy to a monthly dosage of IPTp-SP and advocated for implementation of this policy change [START_REF]WHO | Updated WHO policy recommendation: intermittent preventive treatment of malaria in pregnancy using sulfadoxine-pyrimethamine (IPTp-SP)[END_REF]. Ghana, like other countries adopted and implemented the policy in 2013 providing the opportunity to assess this policy change and its impact based on field observation. Article I of this thesis, which assessed the implementation and impact of the new policy on pregnancy outcomes was the first of such studies in the context of Ghana since its adaptation and implementation by the national health authorities. Here it was shown that IPTp-SP is well implemented in Ghana and uptake of IPTp-SP was high, with over 60% of pregnant women receiving three or more doses of IPTp-SP. Our observation on the implementation and uptake of IPTp-SP in Ghana was not surprising as Ghana has been one of the few countries that have had high coverage of IPTp-SP even before the policy change (52,129). This phenomenon of increased uptake in Ghana can be attributed to an extensive media campaign educating on PAM and its impacts on adverse pregnancy with more emphasis geared towards involvement of their partners in prenatal care. This educational campaign has further been entrenched in the prenatal clinics. This is shown by a study by Boateng and his collogues when they identified early antenatal registration of pregnant women, male partner involvement in antenatal clinic attendance, and frequency of visits to antenatal clinics as the key drivers of IPTp-SP uptake in Ghana (130). Besides these, Ghana was one of the few countries that implemented a three dosage policy before the 2012 policy revision which goes to support the high percentage of pregnant women taking at least three SP doses. Despite the high coverage seen, coverage was still below the WHO target. However, we also noted in our study that IPTp-SP was withheld from some pregnant women due to their glucose 6 phosphate dehydrogenase (G6PD) status without any alternative prevention strategies administered to them. Although this practice was based on a local recommendation from the Ghana Health Service, the withholding of IPTp-SP could be seen in one study site (Maamobi General Hospital) because they had the technical abilities to perform these exclusion tests. This further indicates differences even in the national policy implementation. The observation is also contrary to the WHO policy which does not discriminate IPTp-SP administration on G6PD deficiency. In Africa, some host genetic factors such as, Alpha thalassaemia and sickle cell polymorphisms and other haemoglobinopathies such as G6PD deficiency have all been hypothesized to play a role in malaria protection [START_REF] Nacher | Haematinic treatment of anaemia increases the risk of Plasmodium vivax malaria in pregnancy[END_REF]131). However, some in vitro studies show that this host protective mechanism could also cause hemoglobin hemolysis in the presence of oxidative stress reagents such as sulfonamides. However, there is little evidence supporting this hypothesis even among the few studies done (132). Even more, variation between national and WHO policy means that some pregnant women would be left unprotected to PAM and its adverse effects and further policy change based on scientific evidence and in line with the WHO policy is needed to incorporate this group of women into the IPTp-SP bracket in the context of Ghana. Despite this, uptake of three or more doses was observed in our study to be associated with increased birth weight of more than 0.165kg. This indicates that the new IPTp-SP policy is well implemented in Ghana with corresponding beneficial impact on birth weight. In spite of the observed beneficial clinical efficacy of increased dosage, IPTp-SP is challenged by increased resistance to the chemotherapy of choice (that is SP), especially in southern and eastern Africa. An earlier study by Harrington and his colleagues found a high parasite load carrying the pfdhps resistant allele at codon 581 among pregnant women in Tanzania who had taken IPTp-SP [START_REF] Harrington | Competitive facilitation of drug-resistant Plasmodium falciparum malaria parasites in pregnant women who receive preventive treatment[END_REF]. Others had also found high resistant haplotypes associated with SP resistance among pregnant women with the triple mutant pfdhfr haplotype at near saturation while the pfdhps haplotypes (at codon 436 and 437) ranging from 47% to 90% across sub-Sahara Africa [START_REF] Moussiliou | High rates of parasite recrudescence following intermittent preventive treatment with sulphadoxine-pyrimethamine during pregnancy in Benin[END_REF](133)(134)(135). However, the mutant allele, which is a precedence to increased SP resistance was low in west Africa, although it is the other way round in eastern and southern Africa, ranging from 45%-90% (87) while mutations at codon 581 and 613 varied (136,137). This indicates a compititve development of resistant mutant alleles under continuous drug pressure especially in eastern Africa, but not the case in West Africa. However, in Article-II, we characterized SP resistance molecular markers among parasite isolates obtained from

pregnant women yet to uptake IPTp-SP and among post-IPTp-SP isolates. In this article, we shows that there was a high prevalence of highly mutated phdhfr/pfdhps haplotypes observed among post-treatment isolates compared to first ANC with the quintuple mutation being the dominant haplotypes found which support previous findings of high prevalence of quintuple mutations with near saturation in triple phdhfr mutant haplotypes [START_REF] Moussiliou | High rates of parasite recrudescence following intermittent preventive treatment with sulphadoxine-pyrimethamine during pregnancy in Benin[END_REF]134,135) in west and central Africa. However, the identification of additional mutations at codon 581 and 613 on the pfdhps, particularly among post-IPTp-SP isolates, which previously was rare in west Africa indicates a possible SP treatment selective pressure. This seems to indicate the development and circulation of mutations with high potential of total resistance that could gradually create favorable conditions for the emergence of these phenotypes in the years to come, with the help of the current treatment strategies. The question now is, could the successful implementation of increased IPTp-SP raise fears in this regard? especially in western and central Africa, that have low prevalence of SP resistant mutant parasites in circulation. This, therefore, requires continuous studies to ascertain the efficacy of IPTp-SP and to inform policy on IPTp. Besides parasite resistance, IPTp-SP is also faced with the problem of its application safety window, which excludes the first trimester of pregnancy for safety reasons, while infections occurring at this period are equally harmful to a pregnancy. It has long been suggested that a large percentage of malaria infections carried in early pregnancy reflect infections acquired before conception [START_REF] Walker | A model of parity-dependent immunity to placental malaria[END_REF][START_REF] Berry | Seasonal Dynamics of Malaria in Pregnancy in West Africa: Evidence for Carriage of Infections Acquired Before Pregnancy Until First Contact with Antenatal Care[END_REF][START_REF] Ofori | Etiology of Placental Plasmodium falciparum Malaria in African Women[END_REF]. Evidence from such models are supported by some well-known features of malaria epidemiology including a very high prevalence of malaria infection often observed at first ANC visit regardless of transmission seasons. In Article III, nulligravid women who showed interest in getting pregnant were enrolled and followed until conception and throughout their first pregnancy. This was the first of such studies that demonstrated the feasibility of such kind of longitudinal study. Our study clearly showed that infections occurring during the first trimester of pregnancy were mainly derived from asymptomatic infections, including submicroscopic infections before conception rather than transmission variation as demonstrated by molecular genotyping. This observation demonstrates the difficulty of protecting women from placental infection in the first trimester with the current preventive tools. The use of insecticide-treated nets can not do much for these persistent parasites that are already present in the body. A strategy of intermittent treatment of women with effective and appropriate antimalarials may be imagined in the pre-pregnancy period, but the implementation of such a strategy among adolescent girls or women showing interest in getting pregnant, does not seem obvious. Therefore, a vaccine against PAM would be the ideal strategy choice to offer a better prevention of malaria infection as from the initial moments of pregnancy. VAR2CSA is currently identified as the undeniable ligand that mediates sequestration of P. falciparum infected erythrocytes to placental CSA. This has been based on several epidemiological studies that further showed plasma antibodies against VAR2CSA expressing infected erythrocytes could protect against PAM and its adverse effect, making this protein an attractive target for vaccine development (118-120). However, due to constraints related to large size and structural complexity of the VAR2CSA protein, researchers were led to characterize the binding properties against to CSA and to identify the minimum necessary construct that would be more suitable for vaccine development (118)(119)(120). This resulted in the identification of the N-terminal region of the protein (DBL1x-3×), particularly the ID1-ID2a segment, as the minimal CSA-binding region. This further led to the characterization and development of the PAMVAC vaccine candidate which has entered clinical development (124). However, our understanding of the antibody response on this region of interest remains a necessity and a clear description of IgG isotypes mediating this protection was lacking. In Article IV, we analyzed in a cohort of pregnant women in Benin, the dynamics of IgG acquired against the PAMVAC vaccine construct. This study confirmed the association of high plasma level of antibodies against this area of VAR2CSA and the protection. The study further demonstrated for the first time how different IgG isotypes coordinate to mediate protection against PAM and its adverse effect. Here we clearly observed an association between cytophilic IgG3 and birth weight. Besides, we also observed high levels of IgG4, which was associated with reduced risk of placental infection. This result clearly suggest a coordination of IgG properties in the protective mechanism that VAR2CSA mediates against placental malaria. It suggests a blocking effect of cytoadherence by non-cytophilic antibodies and in particular IgG4. This major result is particularly edifying insofar as it is well known that the placental that houses an allograft should not promote immune reactions that could hinder the good development of the fetus. An inflammatory reaction in this organ might not be ideal, and a vaccine strategy should master this mechanism in order to get the best out of it. It is also clear that the association with birth weight detected with total IgG was also visible on IgG3. From this, it is conceivable that these cytophilic immunoglobulins participate in the clearance mechanism of IEs by recruiting effector cells with opsonophagocytosis potential or complement fixation. This stage could take place away from the placenta, as during filtration in the spleen. Our data, therefore, suggest that any future PAM vaccine should be able to elicit both cytophilic and non-cytophilic IgGs to protect against PAM and its adverse effects. This observation is crucial and further extends our knowledge on anti-VAR2CSA mediating protection by adding an extra. However, to achieve an effective VAR2CSA-based on vaccine that would reproduce this ordered protective mechanism, a determining factor that remains to be overcome is that of having antigens capable of inducing antibodies with these adhesion blocking and parasite killing facilitating properties. A recent study identified a rare sub-variant of P.falciparum that particularly infects multigravid women living in Benin who have already acquired antibodies against VAR2CSA during successive pregnancies. This finding is intriguing due to the fact that these parasites are able to infect pregnant women who had acquired natural immunity to VAR2CSA and also cause high parasitemia. These parasites express a sub-variant of VAR2CSA that was similar to that of a rare isolate identified for the first time in a study in Asia (125).

Further molecular epidemiology studies on the N-terminal region of VAR2CSA on isolates from pregnant women in Benin and Malawi subsequently characterized these subtypes of var2csa as particularly distinct (117). The diversity associated with the differential clinical effects of this variant suggests that an effective vaccine based on VAR2CSA may require a multivalent activity. Article V of this thesis which characterized the antigenic and binding properties of this new variant in comparison with those of FCR3 and 3D7 found a lower binding affinity and strain-specific functional properties of antibodies on the recognition and inhibition of cytoadherence. This strain specific recognition and inhibition properties is what is probably associated to the T2053 infection of pregnant women with acquired natural immunity to VARCSA and the associated high parasite densities among those infected. In addition, the parity dependent recognition of ID1-ID2 recombinant proteins of FCR3 and T2053 by plasmas of pregnant women naturally exposed to P. falciparum in Ghana confirms the dominance of FCR3 variants compared to parasites of T2053 in our study sites. The purpose of vaccination strategies is to induce memory B cells in advance of infection.

Malaria vaccine development is an active research area with enormous challenges.

These challenges stem from the complex life cycle of the parasite as well as the diverse antigens it displays to evade the immune system. However, current advances in molecular biology have identified that an effective malaria vaccine requires (i) multiple components that will induce an effective immune response to the different stages of the malaria infection; (ii) multiple epitopes that are restricted to presentation by different major histocompatibility complex (MHC) molecules to overcome genetic diversity and antigenic variation; and (iii) should be able to inducing more than one type of immune response (138). In the context to VAR2CSA vaccine, the only challenge is antigenic variation among parasite strains. This is the case with the identification of the T2053 strain that preferentially infect multigravid women with high anti-VAR2CSA acquired IgG. However,the pathophysiological effect of this strain compared to that of 3D7 and FCR3 show that parasite isolates harbouring VAR2CSA variants similar to those of 3D7 have been associated with more severe adverse outcomes (125,117). Article V represents the first study to provide a clear perspective on the design of effective multiallele VAR2CSA-based vaccine that would offer a broader spectrum of activity.

VAR2CSA vaccine would be essential in protecting women living in malaria endemic areas from PAM and its adverse effect; however, it appears for this to be successful, it would require a multiallele combination.

  plus les IgG spécifiques induits chez la souris contre les 3 variants montrent des différences majeures dans la reconnaissance croisée. Conclusion : Les travaux réalisés au cours de cette thèse renforcent le fait que le TPI-SP reste confronté à de difficultés majeures notamment sur la couverture du premier trimestre de grossesse où les prévalences d'infections sont très élevées. Le xv développement de mutations parasitaires associées à plus de résistance remet en question l'efficacité de l'utilisation de la SP à terme. De plus, les efforts actuels visant à mettre au point un vaccin contre le paludisme placentaire nécessiteront probablement une combinaison d'allèles pour obtenir un vaccin efficace sur les isolats de terrain. xvi Host laboratory MERIT UMR 261-Mother and child in a tropical environment: pathogens, health system and transition epidemiological Institut de Recherche pour le Développement (IRD) University Paris Descartes Faculty of Pharmacy-Parasitology Laboratory 4, avenue de l'Observatoire xix List of figures

Figure 1 :

 1 Figure 1: P. falciparum-infected erythrocytes sequestration in the placenta ...................

Figure 2 :

 2 Figure 2: Effects of malaria during pregnancy .................................................................

Figure 3 :

 3 Figure 3: Plasmodium falciparum life cycle phases targeted by different antimalarial

Figure 4 :

 4 Figure 4: Malaria infection pattern with age ...................................................................

Figure 5 :

 5 Figure 5: Schematic representation of VAR2CSA constructs ........................................

Figure 1 :

 1 Figure 1: P. falciparum-infected erythrocytes sequestration in the placenta (Sourced

Figure 2 : 1 . 4 . 2

 2142 Figure 2: Effects of malaria during pregnancy. Sources: (adapted from (34))

Figure 3 :

 3 Figure 3: Plasmodium falciparum life cycle phases targeted by different antimalarial

Figure 4 :

 4 Figure 4: Malaria infection pattern with age in a high malaria transmission area

(114, 27 ,

 27 115). Like other VSAs, var2csa is a member of the erythrocyte membrane protein 1 (PfEMP1) family expressed by the var genes, which mediate binding between infected red blood cells (iRBC) to host vasculatures (116). It is a 350 kDa transmembrane protein with a 300 kDa extracellular domain consisting of six Duffybinding-like (DBL) domains and a cysteine-rich interdomain region module, as well as short inter-domain regions Unlike other members of this family, var2csa is unique in terms that it is only expressed in the placenta and thus only binds to chondroitin sulfate A (CSA) expressed on the syncytiotrophoblast.

Figure 5 :

 5 Figure 5: Schematic representation of VAR2CSA constructs. VAR2CSA consists of

(

  121). This finding reshaped the view on placental malaria vaccine and led to the development of two subunit vaccine candidates; (Placental malaria vaccine (PAMVAC) candidate based on the ID1-ID2a of the FCR parasite clone and Priming malaria vaccine (PRIMVAC) which is composed of the DBL1-DBL2 segment of 3D7 parasite clone) (122) that have been shown to be cross-reactive (123) and just completed preliminary clinical trials (phase 1a) (124).

Figure 1 :Effect of drug pressure on promoting the emergence of antimalarial resistant parasites among pregnant women in Ghana Objectives 1 . 2 .

 112 Figure 1: IPTP-SP coverage among delivery women in Maamobi (grey bars) and Kpone on-Sea (stripped bars).

Figure 1 :

 1 Figure 1: Study flow chart. Abbreviations: PCR, polymerase chain reaction; ATS, acidic terminal segment of Pf var gene; pfdhfr, dihydrofolate reductase; pfdhps, dihydropterate synthase and pfk13, kelch13.

Figure 2 :

 2 Figure 2: Location of amino acids associated with pfk13 mutations observed in southern Ghana on the PfK13 propeller tertiary structure. Mutations at positions 481 and 556 are not shown because they are buried in the fold.

Figure 2 82

 2 Figure 2

1. 1 . 1 . 1 . 2

 1112 Ethics Statement Ethical clearance was obtained from the Institutional Ethics Committee of the Faculty of Health Science, Abomey-Calavi University (No. 12/09/2013/CE/FSS/UAC) and the Comité Consultatif de Déontologie et d'Ethique from Institut de Recherche pour le Développement. Written informed consent was obtained from each woman.

Figure 3 .

 3 Figure 3. Prevalence rates of Plasmodium falciparum infections during pregnancy using microscopy or real-time polymerase chain reaction targeting the 18S ribosomal DNA gene (submicroscopic), by parasitological status at enrollment. The number of women followed up in each subgroup is indicated beneath each bar.

  ethics committees: the Comité Consultatif de Déontologie et d'Éthique of IRD in France and the Comité d'Éthique de la Faculté des Science de la Santé, Université d'Abomey Calavi, in Benin. All participants provided written consent.

(

  BSA)-blocked 96-well ELISA plates (Maxisorp, NUNC, Denmark) were incubated overnight at 4ºC with 100 μl/well of test plasma samples in duplicate at dilutions optimized for each subclass (1:200 for total IgG; 1:100 for IgG1 and IgG2 and 1:50 for IgG3 and IgG4) in phosphate-buffered saline (PBS) with 2% BSA. Pools of highly reactive plasma from exposed pregnant Beninese previously characterized on recombinant full-length ectodomain of VAR2CSA (FV2)(11), and non-immune plasma anemia and placental infection in mothers; low birth weight, prematurity, intrauterine growth retardation in newborns. All factors with a p-value <0.20 during univariate analyses were included in the multivariate step. A backward stepwise procedure was used to select factors of the final model. Statistical significance was set at p<0.05.

Figure 1 :

 1 Figure1: IgG subclass profile to ID1-ID2a recombinant protein from pregnant women followed during the course of pregnancy (inclusion, antenatal visits (ANV) and delivery). a The distribution of IgG subclasses. Shown are IgG subclasses antibody levels (median and IQR) in arbitrary units. b IgG subclass seroprevalenc. ). The profile of IgG subclasses clearly shows a cytophilic IgG's (IgG1 and IgG3) are the predominant IgGs while low prevalence of non-cytophilic IgG's (IgG2 and IgG4) were also observed.

  continuous to be the most common and deadliest of the plasmodium species, and infection causes both economic and health challenges. In endemic areas, the disease is mostly a childhood one due to the gradual development of protective immunity, although first-time pregnant women are a notable exception(1). The susceptibility of first-time pregnancy to pregnancy-associated malaria (PAM), irrespective of previously acquired immunity, is attributed to the sequestration of infected erythrocytes in the intervillous spaces of the placenta mediated by their binding to chondroitin sulfate A (CSA). VAR2CSA, a member of the Plasmodium falciparum erythrocyte membrane proteins 1 (PfEMP1), has been identified to be the ligand for CSA and thus a critical factor in the pathology of PAM(2,3). Intermittent preventive treatment of malaria in pregnancy (IPTp) has contributed to minimizing the burden of PAM (4,5), but current chemotherapy strategy used for malaria prevention during pregnancy, sulfadoxine-pyrimethamine (SP) is threatened by high level of resistance(6)(7)(8). However, in P. falciparum malaria-endemic areas, successive pregnancies gain protection against PAM in a parity dependent manner, through antibody-mediated immunity(9,10). Consistent molecular and seroepidemiological studies have facilitated the development of two VAR2CSA-based vaccine candidates. However, Var2CSA

Figure 1 :

 1 Figure 1: Protein expression of ID1-ID2a variant of 3D7 parasite strain. A: A western

Figure 2 :Figure 3 :

 23 Figure 2: Adhesive properties of recombinant ID1-ID2a variants. ID1-ID2a variants

Figure 4 :

 4 Figure 4: Recognition of T2053 ID1-ID2a mice antisera to FCR3 or 3D7 by

Figure 5 :

 5 Figure 5: Naturally acquired antibody recognition profile against ID1-ID2a

Figure 1 Figure 2 3

 12 Figure 1

1.5 Malaria during pregnancy caused by Plasmodium vivax

	(80). This could be due to the increased production of new erythrocytes
	Apart from P. falciparum associated PAM, another species that have also been (reticulocytes) that could be parasitized by P. vivax.
	associated with PAM is P. vivax. However, research on the effects of P. vivax
	associated PAM has lagged behind that of P. falciparum PAM. Several studies
	carried out in Asia and South America have shown the same PAM adverse effects
	caused by P. falciparum. In 1999, Nosten and his colleagues found that first-time
	pregnant women were at an increased risk of P vivax malaria during pregnancy
	(OR= 1.63; CI= 1.37-2.06; p-value<0.001). This was associated with increased risk
	of maternal anemia and low birth weight although in the former among all gravidae.
	However, the effects of P. vivax infection were less striking than those of P
	falciparum although P. vivax first episode reduces the risk of subsequent P.
	falciparum infection (78). Also, other studies in India and Venezuela (79) found
	severe anemia, deficiency of platelets, miscarriages, and preterm deliveries among
	pregnant women with severe P. vivax infections. These studies in conjunction with
	other studies indicate a reduced risk of maternal anemia and birth outcomes among
	pregnant women with P. vivax infection compared to P. falciparum.
	Unlike management of PAM caused by P. falciparum, control of P. vivax caused
	PAM relies only on ITN to prevent vector contact and effective diagnosis and
	treatment with chloroquine in areas chloroquine-susceptible P. vivax are found and
	ACTs where chloroquine resistance P. vivax are predominant, while primaquine is
	also recommended to prevent relapse (1). Contrary to the management of maternal
	anemia among pregnant women infected with P. falciparum, a study by Nacher and
	colleagues suggested an increased risk of P. vivax malaria associated with
	haematinic supplementation among participants in areas where P. vivax is prevalent

1.6 Challenges facing Intermittent preventive treatment

	Despite the current clinical efficacy of IPTp-SP, it is faced with numerous challenges,
	including loss of parasitological efficacy of SP due to resistance, lack of any
	intervention directed to the first trimester of pregnancy, Stock-out of SP at health
	centers, lack of knowledge, poor attitude from health workers and high concentration
	of folic acid utilization.

1.

6.1 Folic acid dosage during pregnancy

1.6.2 Sulfadoxine-Pyrimethamine resistance

	such as the Plasmodium parasites for the de novo biosynthesis of folate required by
	cells for cell division (82). SP exerts its effects through competitive inhibition with
	sulfadoxine mimicking p-aminobenzoic acid (PABA) and compete with it for the
	active site of the enzyme thus preventing the conversion of hydroxy-methyl-
	dihydropterin to dihydropterate needed in a subsequent step in the folate synthesis
	pathway. Also, pyrimethamine mimics pteridine ring of the natural substrate
	dihydrofolate (DHF), thus preventing its NADPH-dependent reduction to
	tetrahydrofolate (THF) necessary for the biosynthesis of thymidylate, purines, and
	methionine (83-85).
	In vivo SP resistance is known to be due to point mutations within the gene that
	encode the enzymes pfdhfr and pfdhps, resulting in amino acid substitution at
	specific positions with these enzymes. These amino acid substitutions occur at
	codons 51 (N51I), 59 (C59R), 108 (S108N) and 164 (L164I) for pfdhfr and codons
	436 (S436A/F), 437 (A437G), 540 (K540E), 581 (A581G) and 613 (A613S/T) for
	pfdhps. Over the past decades, parasite resistance to SP in vivo has been mostly
	associated with triple dhfr mutations (51, 59, and 108) coupled with a double dhps
	mutations (437 and 540) (quintuple) which are saturated mostly in Eastern Africa
	Sulfadoxine and pyrimethamine are antifolate chemotherapies; a class of antimalaria
	drugs that exert their effect by synergistically inhibiting dihydropteriate synthase
	(dhps) and the activity of the bifunctional dihydrofolate reductase (dhfr)/ thymidylate
	synthase enzyme. These enzymes are unique to plants and most microorganisms

Table 1

 1

	)(41,97). However, a policy change was initiated after
	the adoption and implementation of the strategic framework for malaria prevention
	and control during pregnancy in the Africa region to safeguard SP; the drug of choice
	for IPTp. This policy change requires SP (fansidar) not to be available/sold outside
	health institutions and should only be administered as IPTp to stall SP resistance
	development. A recent finding shows high plasma level of SP among pregnant
	women before their commencement of IPTp-SP in Ghana which could indicate the
	use of this drug before health care visit (Quakyei et al, under review). This
	phenomenon is also observed in Kenya where many antimalarial prescription-only
	medicines are sold over the counter raising fears among health practitioners (98).
	Therefore, in the short term, judicious application of SP should be enforced to help
	reduce the rate of SP resistance development thereby prolonging the clinical and
	parasitological efficacy of SP for IPTp.
	Over the past decades, as SP resistance increases and spreads, several studies
	have looked at alternative drugs (either as monotherapy or combination therapy) to
	SP for use in IPTp. Mefloquine was one of the promising candidates as it had
	several desirable characteristics and also had good clinical and parasitological
	efficacy among pregnant women when used as weekly prophylaxis (99). However,
	its intolerability by pregnant women in clinical trials (100,101) informed a WHO
	evidence review committee to reject it as a replacement for SP in IPTp (102). Other
	studies exploited the use of azithromycin-chloroquine or azithromycin-SP

Table 1 : Antimalaria policy in selected countries before the implementation of IPTp-SP Source: (adapted from (99)) 1.8 Malaria vaccine development

 1

	The quest for an effective vaccine against malaria remains a global priority. Even
	though classical vaccine design strategies have advanced, and although this has
	been successful for some viral (smallpox and rotavirus) and bacterial pathogens
	(pneumococcus) (107), little success has been achieved for Plasmodium falciparum.
	This unsuccessfulness still hinger on the parasite complex genomic and life cycle.
	However, extensive progress has been made over the past decades since the first
	unsuccessful recombinant peptide-based malaria vaccine development by

  Sea. At Maamobi, we detected sulfadoxine among 306 (75.4%) of peripheral plasma samples at median concentration of 3296mg/ml (IQR: 631.8 -9748) compared to 265 (72.6%) cord plasma samples at median concentration of 2626 mg/ml (IQR: 625.5 -8403).

	Pyrimethamine was detected in peripheral plasma of 117 (28.8%) women at median
	concentration of 18ng/ml (IQR: 12 -32), and 49 (13.4%) cord plasma samples at
	concentration of 25ng/ml (IQR: 13 -25). At Kpone on-Sea, we detected sulfadoxine residual
	levels among 114 (87.7%) peripheral plasma at median concentration of 10945mg/ml (IQR:
	3169 -27328) and 113 (87.7%) cord samples at median concentration of 10841 mg/ml (IQR:
	4062 -27798). Besides, we detected pyrimethamine residual levels among 77 (59.2%)

Assays were performed in 406 peripheral and 365 cord plasma samples from Maamobi compared to 130 paired peripheral and cord plasma samples from Kpone onperipheral plasma at median concentration of 27 ng/ml (IQR: 13.3 -80.5) and 55 (42.3%) cord samples at median concentration of 50 ng/ml (IQR: 20 -120).

Table 1 :

 1 Study population characteristics

		1st ANC visit		Delivery
	Characteristics		Kpone on-Sea		Kpone on-Sea
		Maamobi (n=490)		Maamobi (n=635)
			(n= 497)		(n= 300)
	Age #	27.28±5.98	27.27±5.83	28.13±5.57	26.67±5.32
	Marital Status †
	Single	123 (25.2%)	37 (7.4%)	141 (22.2%)	44 (14.7%)
	Co-habiting	46 (9.4%)	141 (28.4%)	22 (3.5%)	67 (22.3%)
	Married	320 (65.4%)	319 (64.2%)	460 (72.4%)	189 (63%)
	Educational level †
	None	48 (9.9%)	44 (8.9%)	68 (10.7%)	31 (10.3%)
	Basic education	285 (59.0%)	337 (67.8%)	249 (39.2%)	197 (65.7%)
	Middle education	103 (21.3%)	87 (17.5%)	147 (23.2%)	53 (17.7%)
	Higer education	47 (9.7%)	29 (5.8%)	62 (9.8%)	19 (6.3%)
	Employment Status †
	Yes	389 (79.4%)	375 (75.5%)	496 (78.1%)	232 (77.3%)
	No	101 (20.6%)	122 (24.5%)	138 (21.9%)	68 (22.7%)
	Gravidy/Parity †
	Primigravidae	126 (25.7%)	123 (24.7%)	121 (19.1%)	75 (25%)
	Pf. infection diagnosed
	By 18SqPCR	16 (12.7%)	13 (10.6%)	5 (4.1%)	2 (2.7%)
	By ATSqPCR	50 (39.7%)	58 (47.1%)	35 (28.9%)	10 (13.3%)
	Multigravidae	364 (74.3%)	374 (75.3%)	513 (80.9%)	225 (75%)
	Pf. infection diagnosed
	By 18SqPCR	30 (8.2%)	31 (8.3%)	23 (4.5%)	7 (3.1%)
	By ATSqPCR	159 (43.7%)	156 (41.7%)	45 (8.8%)	16 (7.1%)
	Gestational age at first ANC #	18.4±6.89	17.9±7.13	18.9±7.29	18.3±6.95
	Bednet possession †
	Yes	293 (59.8%)	214(43.1%)	498 (78.4%)	272 (90.7%)
	No	197 (40.2%)	283 (56.9%)	137 (21.6%)	28 (9.3%)
	Bednet usage †
	Always	93 (31.7%)	141 (65.9%)	187 (37.5%)	141 (51.8%)

#Mean ± SD; † n (%); *p-value< 0.005; G6PD defect comprises partial and full deficit, ND not done

Table 2 :

 2 P.falciparum prevalence among different sample types by both 18SqPCR and

	ATSqPCR
		1st ANC		Delivery
		Maamobi	Kpone on-	Maamobi	Kpone on-
	Characteristics	(n=490)	Sea (n= 497)	(n=634)	Sea (n= 300)
	Peripheral
	infection*
	By 18SqPCR	46(9.4%)	44(8.9%)	28(4.4%)	9(3.0%)
	By ATSqPCR	207(42.2%)	214(43.1%)	80(12.6%)	26(8.7%)
	Parasite density#
	By 18SqPCR	21460±42907	27484±74382 27847±130059 21943±43029
	Placental infection
	By 18SqPCR			16(2.5%)	5(1.7%)
	by ATSqPCR			44(6.9%)	11(3.7%)

* n (%); #Mean ± SD;

Table 3 :

 3 The concordance and discordance between infections detected targeting the 18 SqPCR and the ATS qPCR

			ATS qPCR
	18SqPCR	Positive	Negative	Row total
	Positive	134	1	135
	Negative	432	2253	2685
	Column total	566	2254	2820

Table 4 :

 4 Relationship between P. falciparum infections detected by ATSqRT-PCR and IPTp-SP a uptake using a multivariate logistic regression

	Risk factor

* Plasmodium falciparum

Table 5 :

 5 Relationship between SP uptake and birth weight by multivariate linear regression

	95%	confidence
	Coefficients (Kg)	p-value
	interval

a SP uptake of less than 3 doses (1 or 2 SP uptake) reflecting the old IPTp policy recommendation; b SP uptake of 3 or more doses reflecting the 2012 recommendation.

Table 6 :

 6 Relationship between IPTp-SP uptake and residual SP a level measured in the blood at delivery

	SP measurement	OR b	95% confidence interval	P value
	Primigravidae	0.67	(0.32; 1.41)	0.29
	IPTp uptake
	1 or 2 IPTp-SP doses *	18.9	(6.5; 54.6)	<0.0001
	≥3 IPTp-sSP doses taken #	32.3	(13.3; 78.4)	<0.0001
	Site	0.9	(0.4; 1.8)	0.8

a sulfadoxine as a proxy for SP categorised into binary variables (either high or low SP level) base on the median; b Odds-Ratio, *Participants that reported to have received 1 or 2 SP doses, #Participants that reported to have received ≥ 3 SP doses. The reference classes are the multigravidae, the women who reported no dose of SP taken, and Maamobi site.

  .6% (153/223), 63.2% (141/223), and 46.2% (103/223) of samples were successfully amplified, sequenced and genotyped respectively for the pfdhfr, pfdhps genes and the pfk13 propeller-encoding gene fragment.

	associated with sulfadoxine resistance, the prevalence of mutant alleles was high at codons
	436 (S436A/F, 95.0%, 134/141) and 437 (A437G, 96.4%, 133/141), but very low at codon
	540 (K540E, 0.7%; 1/141). Mutations at codons 581 (A581G) and 613 (A613S/T) were
	respectively found in 12.8% (18/141) and 17.7% (25/141) of the isolates (Table

At ANC, 63.4% (97/153), 65.2% (92/141), and 50.5% (52/103) isolates were successfully genotyped respectively for pfdhfr, pfdhps and pfk13. At delivery, 37.2% (57/153), 32.6% (46/141) and 49.5% (51/103) were also genotyped for pfdhfr, pfdhps, and pfk13 respectively.

Prevalence of pfdhfr and pfdhps mutations

Mutant pfdhfr alleles were predominant at codons 108 (S108N, 93.5%, 112/153), 59 (C59R, 90.8%, 111/153) and 51 (N51I, 85%, 130/153; Table

1a

). The high prevalence of these mutant pfdhfr alleles was observed at both time points (Table

1b

). In the pfdhps gene

  further investigations are needed to check whether they confer survival advantage to parasites pressured by ART. Importantly, one Singaporean returning from Ghana presented

with a P. falciparum infection exhibiting the N458D mutation and was not effectively cured by

(31)

. This findings in addition with those reported by

Ocan et

al. (2016) from Uganda (32), Bayih et al. (2016) in northwest Ethiopia (33) and Ouattara et al. (2015) from Mali

Table 1a :

 1a Prevalence of pfdhfr, pfdhps, and pfk13 SNPs among pregnant women in study isolates

				Table1b
			first ANC (n = 99)			Delivery (n = 57)
	Gene Gene	SNP	SNP * Wild type	Wild type, n (%) mutations	Mutation, n (%) Mixed type Wild type	Mixed type n (%) mutations	Mixed type
	pfdhfr # (n = 153) pfdhfr N51I	N51I C59R 15 (15.2%)	23 (15.0%) 14 (9.2%) 82 (82.2%)	130 (85.0%) 111 (90.8%) 0 (0%) 8 (13.6%)	0 (0%) 0 (0%) 48 (81.4%)	0 (0%)
		C59R	S108N 8 (8.1%)	10 (6.5%) 89 (89.9%)	112 (93.5%) 0 (0%) 6 (10.2%)	0 (0%) 50 (84.8%)	0 (0%)
		S108N	I164L 5 (5.1%)	153 (100%) 92 (92.9%)	0 (0%)	0 (0%) 5 (8.5%)	0 (0%) 51 (86.4%)	0 (0%)
	pfdhps a (n = 141) pfdhps S436A/F	S436A/F A437G 1 (1.0%)	2 (1.4%) 5 (3.6%) 91 (91.9%)	134(95.0%) 133 (96.4%) (2.0%) 2 1 (1.8%)	5 (3.6%) 0 (0%) 43 (75.4%)	3 (5.3%)
	A437G	K540E 2 (2.0%)	140 (99.3%) 90 (90.9%)	0 (0%)	1 (0.7%) 0 (0.0%)	0 (0%) 46 (80.7%)	0 (0%)
		K540E	A581G A613S/T 91 (91.9%)	123 (87.2%) 116 (82.63%) 1 (1.0%)	18 (12.8%) 25 (17.7%) 0 (0%) 44 (77.2%)	0 (0%) 0 (0%) 0 (0.0%)	0 (0%)
	pfk13 b (n = 103) A581G	E455G 83 (83.8%)	101 (98.1%) 9 (9.1%)	0 (0%)	2 (1.9%) 35 (61.4%)	9 (15.8%) 0 (0%)
	A613S/T	N458D A481C 77 (77.8%)	101 (98.1%) 102 (99.0%) 15 (15.2%)	0 (0%)	2 (1.9%) 1 (1.0%) 34 (59.6%)	10 (17.5%)	0 (0%)
			T535A	102 (99.0%)			1 (1.0%)
			Y616S	102 (99.0%)			1 (1.0%)
			L618V	101 (98.1%)			2 (1.9%)
			A621G	102 (99.0%)			1 (1.0%)
			L663I	102 (99.0%)			1 (1.0%)
			N672I	101 (98.1%)			2 (1.9%)

Table 2 :

 2 Prevalence of pfdhfr and pfdhps haplotypes

	gene	category	haplotype	n (%)
	* pfdhfr (n = 153)	wild type	NCSI	10 (6.5%)
		single	NCNI	(0.7%)
		double	ICNI	(2.0%)
			NRNI	12 (7.8%)
		triple	IRNI	(79.7%)
	# pfdhps (n = 141)	single	A/FAKAA	(1.4%)
			SGKAA	(2.1%)
		double	A/FGKAA	101 (71.6%)
		triple	A/FGEAA	(0.7%)
			A/FGKGA	(2.8%)
			A/FGKAS/T	11 (7.8%)
		quadruple	A/FGKGS/T	14 (9.9%)
		Mixed type	S/AAKAA	(1.4%)
			S/AGKAA	(2.1%)

* dihydrofolate reductase (pfdhfr), # dihydropterate synthase (pfdhps)

Table 3 :

 3 Combined pfdhfr/pfdhps haplotypes based on sample time point and study site

				First ANC	Delivery
	Gene	category	Haplotype	(n = 82)	(n = 44)
	pfdhfr/pfdhps
	n = 126	wild type	NCSI/SAKAA	1 (1.2%)	0 (0.0%)
		double	NCSI-A/FGKAA	3 (3.7%)	4 (9.1%)
		triple	NCNI-A/FGKAA	1 (1.2%)	0 (0.0%)
		quadruple	ICNI-A/FGKAA	2 (2.4%)	0 (0.0%)
			NRNI-A/FGKAA	8 (9.8%)	1 (2.3%)
			IRNI-A/FAKAA	1 (1.2%)	0 (0.0%)
		quintuple	IRNI-A/FGKAA	52 (63.4%)	28 (63.6%)
		sixtuple	IRNI-A/FGEAA	1 (1.2%)	0 (0.0%)
			IRNI-A/FGKAS/T	8 (9.8%)	3 (6.8%)
		septuple	IRNI-A/FGKGS/T	5 (6.1%)	8 (18.2%)

Table 3 . Genotyping of Recurrent Infections From Women Infected Before Pregnancy a

 3

			Infections Before			Infections During Pregnancy
			Pregnancy
						Parasite Density,		Persistent
	Woman	Infection Type	Parasite Density, Parasites/µL b	Genotypes, No.	Infection Type	Parasites/µL b	Genotypes, No.	or Reinfection
	A021	Microscopic	249	4	Submicroscopic	0	3	Persistent
	A023	Microscopic	40	5	Microscopic	26 280	5	Persistent
	A055	Microscopic	662	4	Microscopic	1059	4	Persistent
	A071	Microscopic	268	4	Microscopic	177	3	Reinfection
	A135	Microscopic	168	2	Microscopic	1625	2	Reinfection
	B037	Microscopic	343	5	Microscopic	2235	6	Persistent
	B093	Microscopic	45	4	Microscopic	1089	3	Persistent
	A069	Submicroscopic	0	6	Microscopic	258	2	Persistent
	A075	Submicroscopic	0	2	Microscopic	1656	2	Persistent
	A104	Submicroscopic	0	5	Microscopic	160	1	Reinfection
	A111	Submicroscopic	0	4	Microscopic	12	3	Persistent
	A119	Submicroscopic	0	7	Microscopic	222	6	Persistent
	B055	Submicroscopic	0	4	Microscopic	15	3	Reinfection
	B067	Submicroscopic	0	5	Microscopic	1493	5	Persistent
	B088	Submicroscopic	0	5	Microscopic	4910	5	Persistent
	B099	Submicroscopic	0	6	Microscopic	159	3	Persistent
	B122	Submicroscopic	0	4	Microscopic	12	2	Persistent
	B128	Submicroscopic	0	2	Microscopic	3510	4	Reinfection
	a

Table 1 :

 1 Characteristics of the population

	Mothers

BS: Blood smear; SGA: small for gestational age

Table 3 :

 3 Factors associated with antibody level

	Covariates	Total IgG				IgG1				IgG2				IgG3				IgG4
		Univariate		Multivariate		Univariate		Multivariate		Univariate		Multivariate		Univariate		Multivariate		Univariate		Multivariate
				Adjusted				Adjusted				Adjusted				Adjusted				Adjusted
		β	P	β	p	β	p	β	p	β	p	β	p	β	p	β	p	Β	P	β	P
	ANVs		0.001		0.005		<10-3		0.015
	Inclusion	-		-		-		-		-		-		-				-
	ANV1	-0.95		-0.78		-2.61		-2.26		-0.15		-0.06		-1.03				-3.81	0.32
	ANV2	-2.77		-2.31		-2.56		-1.79		-2.4	<10-3	-2.33	<10-3	-2.79	0.006			-1.27
	ANV3	-2.51		-1.99		-4.01		-3.12		-4.76		-3.59		-4.18				-2.3
	Delivery	-1.72		-2.19		1.95		0.85		-4.41		-4.47		-1.46				-3.57
	Primiparity																	-
	No	-		-		-				-				-				-0.08	0.98
	Yes	-9.07	0.002	-9.9	0.001	-3.29	0.49			-7.15	0.09			2.17	0.55
	Malaria
	infection *										0.23
	0	-	<10-3	-	<10-3	-	<10-3	-	<10-3	-				-	<10-3	-	<10-3	-
	1	5.18		4.46		6.16		4.92		2.21				8.12		7.94		-1.81	0.7
	2	7.73		7.93		12.92		12.37		0.93				10.08		10.12		-1.54
	≥3	13.56		14.3		7.66		5.44		-0.35				2.97		2.93		-13.56
	Parasite																	-
	density	0.0002	<10-3	0.0001	0.028	0.0004	<10-3			0.0001	0.09	0.0001	0.03	0.00004	0.59			0.0001	0.33
		*(number of documented blood smear positive during follow-up)

Acknowledgement

We are grateful to all the women who participated in the study. We thank the staff of the different study sites for their contribution to the collection of samples and Mr. Emmanuel Kakra Dickson for his technical assistance.

BT was supported by a joint ARTS PhD fellowship from IRD and SCAC fellowship from the French Embassy in Ghana.

The project was funded by the Canal 1 grant from Initiative 5%, Expertise France: Grant No. 14SANIN163

Acknowledgement: We are grateful to all the women who participated in the study. We thank the staff of the different study sites for their contribution to the collection of samples and Mr. Emmanuel Kakra Dickson for his technical assistance.

BT was supported by a joint ARTS PhD fellowship from IRD and SCAC fellowship from the French Embassy in Ghana.

The project was funded by the Canal 1 grant from Initiative 5%, Expertise France: Grant No.

14SANIN163

Acknowledgments. This study is part of the PlacMalVac collaborative project, "Clinical Development of a VAR2CSA-Based Placental Malaria Vaccine." We thank Parfait Houngbegnon for assistance in data analysis. Financial support. This study was supported by the European Seventh Framework Programme (contract 304815). Potential conflicts of interest. All authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.

Declaration of interests:

No competing financial and/or non-financial interests in relation to the work described.

Ethics statement

All experimental protocols were approved by the Institutional Review Board (IRB) of the Noguchi Memorial Institute for Medical Research (NMIMR) and the Ethics Review Committee of the Ghana Health Service (GHS). Written informed consent was obtained from each participant before they were admitted into the study. All methods were carried out in accordance with the relevant guidelines and regulations.

Data and biological sample collection

Standardised questionnaires and checklists were used to collect demographic, clinical and ultrasound data for the cohort at the first ANC. As standard practice, pregnant women attending ANC in these two centres are given IPTp-SP under direct observation, and the health staff make entries into their booklets after this directly observed therapy (DOT). Three

(3) ml of peripheral blood were collected into EDTA vacutainer tubes before administration of IPTp-SP. Malaria screening by light microscopy and haemoglobin (Hb) level determinations by Hemocue® were performed.

For the cohort at delivery, standardised questionnaires and checklists were also used to collect demographic and clinical data. Information on IPTp-SP uptake was obtained from participants' ANC booklets. Three (3) ml of peripheral blood and 3 ml of cord blood were collected into separate EDTA vacutainer tubes, and peripheral Hb level measured. Thick and thin blood smears were made for microscopic detection of Plasmodium. A piece of delivered placenta from the mother's side was cut and blots made from it.

All blood samples were centrifuged, and the separated components (plasma and red blood cell pellets) were stored at -80 and -20 ˚C respectively for further analysis.

Parasitological determinations by microscopy and real-time PCR assay

Author contributions NTN, IQ, MFO, JF, KAK and AKA conceived and designed the study; BT, NC, AL and GQA carried out the sample collections; BT, GE and PH performed the molecular and SP level measurements; BT, MC, PD, and GC performed the statistical/data analysis; BT, KAK and NTN wrote the paper.

amodiaquine, lumefantrine, primaquine, artemisinin and quinine. Homemade and external controls obtained from the worldwide antimalarial resistance network (WWARN) were used as controls.

Statistical analysis

All data were analyzed with R programming. Chi-squared test was used to determine the association between the SNPs of the pfdhfr and pfdhps genes and the number of dosage of SP taken. Association between mutations observed and the level of SP in the peripheral plasma were tested using welch t-test or logistic regression where appropriate. P-values less than 0.05 indicated significance.

Declaration of interests: No conflict.

Author contributions: NTN, IQ, MFO, KAK and AKA conceived and designed the study; BT, NC, and AM carried out the sample collections; BT performed the parasite genotyping; BT and PH carried out the SP level measurements; BT, RT and BA performed the sequence analysis and statistical analysis; BT, RC, RT and NTN wrote the paper; JC and PD reviewed the MS. 2). Each loop connects two strands: for example, DA loop connects the strand D with the strand A of the next blade. c A position is supposed to be exposed to the solvent when it exhibited relative solvent accessibility ≥ 16%. d Values correspond to the folding free energy (ΔΔG), expressed in kcal/mol. Negative and positive values suggest destabilizing and stabilizing effects respectively.

Clinical Infectious Diseases

Persistent Plasmodium falciparum Infection in Women With an Intent to Become Pregnant as a Risk Factor for Pregnancy-associated Malaria Nicaise Tuikue Ndam, 1,2 Bernard Tornyigah, 1,2 Akpéyédjé Yannelle Dossou, 3 Guillaume Escriou, 1 Morten A. Nielsen, 4,5 Ali Salanti, 4,5 Saadou Issifou, 3 Achille Massougbodji, 3 Jean-Philippe Chippaux, 1,3 and Philippe Deloron 1 Background. Pregnant women are more susceptible to Plasmodium falciparum than before pregnancy, and infection has consequences for both mother and offspring. The World Health Organization recommends that pregnant woman in areas of transmission receive intermittent preventive treatment (IPTp) starting in the second trimester. Consequently, women are not protected during the first trimester, although P. falciparum infections are both frequent and harmful.

Methods.

A cohort of nulligravid women was followed up during subsequent pregnancy. Malaria was diagnosed by means of microscopy and polymerase chain reaction. Parasites were genotyped at polymorphic loci.

Results.

Among 275 nulligravidae enrolled, 68 women became pregnant and were followed up during pregnancy. Before pregnancy, P. falciparum prevalence rates were 15% by microscopy and 66% by polymerase chain reaction. Microscopic infection rates increased to 29% until IPTp administration, and their density increased by 20-fold. Conversely, submicroscopic infection rates decreased. After IPTp administration, all types of infections decreased, but they increased again late in pregnancy. The risk of infection during pregnancy was higher in women with a microscopic (odds ratio, 6.5; P = .047) or submicroscopic (3.06; P = .05) infection before pregnancy and was not related to the season of occurrence. Most infections during pregnancy were persistent infections acquired before pregnancy.

Conclusions. Microscopic and submicroscopic malaria infections were frequent in nulligravid women from south Benin. During the first trimester of pregnancy, microscopic infections were more frequent, with a higher parasite density, and mainly derived from parasites infecting the woman before conception. Preventive strategies targeting nonpregnant women with a desire for conception need to be designed.

Keywords.

malaria; pregnancy; genotypes; preconception.

Despite huge achievements in malaria control programs, Plasmodium falciparum malaria remains a major public health problem in most tropical endemic areas. Pregnant women are particularly concerned, because they become more susceptible to P. falciparum infection than before their pregnancy, and because the infection affects both mother and child. Therefore, the World Health Organization (WHO) recommends that pregnant women in areas of moderate to high malaria transmission receive intermittent preventive treatment (IPTp).

IPTp is given as a full therapeutic course of sulfadoxinepyrimethamine (SP) at each scheduled antenatal care visit, during pregnancy were consistently associated with higher birth weights, whilst high levels of IgG4 were associated with a reduced risk of placental infections. This suggests that protective anti-VAR2CSA IgG responses are coordinated between both cytophilic and non-cytophilic antibodies.

from unexposed French volunteers, were used on each plate as internal controls.

Reactions were developed with goat anti-human IgG conjugated to horseradish peroxidase (SIGMA, France) followed by the substrate TMB (SIGMA, France). The color reaction was stopped by the addition of 0.2M H2SO4 and optical densities (ODs)

were read at 450 nm. In between all incubation steps, plates were washed 3 times with PBS, pH 7.4, containing 0.1% Tween 20, using an automated plate washer.

ELISA for the subclasses followed the same procedure except using mouse antihuman IgG subclasses Fc secondary antibody conjugated to HRP (MH1715, MH1722, MH1732, or MH1742, all from Invitrogen, France). All reagents were used at predetermined concentrations and optical densities (OD) generated from the antibody measurements were converted to arbitrary units (AUs), as described (17).

Briefly, AUs were calculated as follows:

Statistical analysis

The outcome variable was the level of IgG antibodies in peripheral maternal blood.

Five principal outcome variables were created: the level of total IgG, the levels of

Article V

The critical antigenic diversity of var2csa for optimal vaccine development Objective 1. To investigated the the antigenic and binding properties of ID1-ID2a construct of an African isolate of T2053, in comparison with those of the FCR3 and 3D7 variants which are more common.

Key findings

 Preferential binding of the ID1-ID2a variant of T2053, FCR3 and 3D7 to decorin  Low recognition of T2053 mouse antiserum to either ID1-ID2a variants of FCR3 and 3D7 recombinant proteins or FCR3 and 3D7 parasite lines expressing VAR2CSA  High reactivity of T2053 recombinant protein among multigravida women in

Ghana

Abstract

In malaria-endemic areas, protective immunity to placental malaria is acquired over successive pregnancies through anti-VAR2CSA antibodies. However, recent identification of a Plasmodium falciparum isolates expressing a sub-variant of VAR2CSA in exposed multigravid women suggests that a successful VAR2CSA-based vaccine may require a cocktail of variants. We have identified and established in culture a field isolate expressing this sub-variant and assessed the functional and antigenic properties of the ID1-ID2a region of VAR2CSA expressed by this variant.

Result: The ID1-ID2a recombinant protein for this variant showed a lower affinity for CSA compared to similar constructs of the FCR3 and 3D7 variants. Although their recognition by the plasmas of exposed African pregnant women was equally less important, the specific antibodies induced in mice showed very strain-specific functional properties on the recognition and inhibition of cytoadherence.

Conclusion:

This study provides a clear perspective on the design of effective multiallele VAR2CSA-based vaccine that would offer a broader spectrum of activity.

infected with parasites expressing var2csa and associating high parasite densities is an intriguing observation. This observation is all the more intriguing because these parasites express a sub-variant of var2csa that is similar to that of a rare isolate identified for the first time in a study in Asia (11). Molecular epidemiology studies on the N-terminal region of var2csa on isolates from pregnant women in Benin and Malawi subsequently characterized these subtypes of var2csa as particularly distinct (12). In these two African malaria-endemic settings, parasite isolates harboring VAR2CSA variants similar to those of 3D7 have been associated with more severe adverse outcomes, supporting the differential effects of infection with placental parasite strains.

The diversity associated with the differential clinical effects suggests that an effective vaccine based on VAR2CSA may require multivalent activity (12). In this study, we investigated the antigenic and binding properties of ID1-ID2a construct of an African isolate of this type (T2053), in comparison with those of the FCR3 and 3D7 variants which are more common.

Methods

Ethics statement

Ethical clearance on all human samples used were approved by either the Comite´

Identification of T2053 variant

Based on the VAR2CSA sequences available online and those generated in our previous work (11,12), we performed alignments of the ID1 region and defined specific qPCR primers (Fw AATCGGCGAAAACGGATGGA; Rv TCTCAAGASAGGGGAGCAGTT), capable of discriminating VAR2CSA variants of FCR3, 3D7 and those of new clades types mostly found in multigravidae (11).

Screening our banks of frozen field isolates allowed us to identify a monoclonal isolate (T2053) whose sequence was subsequently validated by sequencing.

P. falciparum in vitro culture and VAR2CSA selection

P. falciparum FCR3, 3D7, and T2053 parasite lines were cultured under standard conditions in O+ RBC in RPMI 1640 containing L-glutamine (Invitrogen France) supplemented with 5% Albumax I, 5% Human plasma, 1× hypoxanthin, and 20 μg/mL gentamicin (13). Parasitemia was routinely monitored by a thin blood smear after Giemsa staining (Sigma-Aldrich). VAR2CSA-expressing IEs were selected on choriocarcinoma cell line BeWo as described ( 14) after repeated panning. The expression of VAR2CSA on the surface of the IEs was verified by flow cytometry with a highly cross-reactive DBL1-6FCR3-rabbit IgG.

Protein expression, purification, and evaluation

The ID1-ID2a variants of VAR2CSA from 3D7, FCR3 and the T2053 parasite lines were cloned into an E. coli SHuffle T7 expression strain vector upstream of a histidine tag in the C-terminal end of each construct (New England Biolabs). The transformed vector cell lines were grown overnight at 37ºC in LB medium with 30 mg/ml of kanamycin.

Protein expression was carried out overnight at 16ºC with 0.3mM Isopropyl β-D-1thiogalactopyranoside (IPTG). All purification steps were performed at 4 °C. Bacterial pellets were lysed with lysozyme and by sonication in lysis buffer with protease inhibitor cocktail tablet from sigma-Aldrich. (lysis buffer: 20 mM Tris-HCl pH 8, 500 mM NaCl, DTT). The lysates were cleared by centrifugation (20000 rpm at 4 °C for 30 min), and the soluble fraction was affinity purified using His-Trap on Aktä Pure System (GE Healthcare). Gel filtration of the eluted fractions was further carried out on Superdex 75 16/60 column (GE Healthcare) using lysis buffer. The fractions corresponding to the correct protein size were analyzed on a 10% SDS PAGE to confirm the size of the eluted protein.

ELISA binding assay

The capacity for the ID1-ID2a variants of T2053, FCR3 and 3D7 were assessed by indirect enzyme linked-immunosorbent assay. A 96-wells ELISA plate was coated overnight at 4˚C with 0.5 μg/mL decorin in PBS (Gibco, [pH 7.2]). Wells were blocked with PBS 1% BSA dilution buffer for) 1 h at 37˚C. A two-fold serial dilution of the three recombinant proteins in dilution buffer was added from 4μg/ml to 0.125μg/ml and incubated for two hours at room temperature. The plates were developed with a PentaHIS horseradish peroxidase (HRP)-conjugated antibody (Qiagen, #1014992) followed by the substrate TMB. The color reaction was stopped by the addition of 0.2MH2SO4, and optical densities (ODs) were subsequently read at 450 nm using a 96well ELISA plate reader. In between all incubation steps, plates were washed four times with PBS, pH 7.4, 0.05% Tween-20 using an automated plate washer.

Mice immunization with T2053 ID1-ID2a recombinant variant and antibody level measurements

Mice immunizations with the T2053 were carried out, as described by (15). Briefly, 6week-old Swiss female mice were anesthetized by intraperitoneal injection of 0.3 mL of a cocktail of ketamine (8.66 mg/mL) and xylazine (0.27 mg/mL) in 150 mM sodium chloride. Mice immunizations were performed by intraperitoneal injection of ID1-ID2a variant of T2056 recombinant protein formulated in Alugel mix. Animals were immunized at days 0, 15, and 45, and antisera were collected at days 0 and 56. IgG serum titres were determined by enzyme-linked immunosorbent assay (ELISA) tests as described by (16) with TMB as the substrate and substrate reaction measured at 450nM.

IgG Preparation and evaluation on the recognition of the native protein

Total IgG from final bleed mouse was manually purified on a Hi-Trap Protein G High Pressure (HP) column according to the manufacturer's recommendations (GE Healthcare). Induced antibody reactivity to FCR3 and 3D7 laboratory parasite lines repeatedly panned to express VAR2CSA were analyzed using a FACS Calibur flow cytometry as previously described (17). Briefly, late-stage infected erythrocytes expressing VAR2CSA were labelled with ethidium bromide and subsequently exposed to purified anti-T2053 IgG or purified IgG pool from multigravid women. All VAR2CSAantibody reactivity was further detected by FITC-conjugated anti-mice IgG.

Competition ELISA

Before performing the competition ELISA, standard ELISA was used to titrate serum dilutions and antigen coating concentration as well as optimization of secondary antibody concentrations and incubation times for the substrate. We further established the optimal serum dilution of pooled anti-T2053 sera (Plasma pool from D56) required to reach the saturation point. Additionally, we titrated the concentration of the competitor antigen to determine the concentration required to saturate antibody binding thoroughly and achieve maximum inhibition. A concentration of 2ug/mL of competition antigen was well above the saturation threshold and was used in all competition ELISAs as described by (18). Briefly, for each of the three antigens used as coating antigen, the same antigen and any of the other antigen combinations were added as the competitor.

Anti-T2053 sera /competitor mix was allowed to pre-incubated for 30min at room temperature before adding to the plate and incubated for 2 hours. To determine IgG antibody bound, HRP conjugated anti-mouse IgG used to develop the plate, followed by substrate addition of TMB. The reaction was stopped with 0.2MH2SO4, and the OD read at 450 nm using a 96-well ELISA plate reader.

Characterization of the recognition of expressed proteins by plasma of pregnant

women naturally exposed to P. falciparum ID1-ID2a variants of FCR3 and T2053-specific IgG were measured by an indirect ELISA as previously described (16,19) Specific IgG present in the plasma bind to the ID1-ID2a variant of FCR3 or T2053 coated onto the microplate wells and detected by horseradish peroxidase (HRP) conjugated goat anti-human IgG antibody. HRP activity was measured by color conversion of the substrate TMB, and the absorbance was measured at 450nM after stopping the reaction with 0.2M sulfuric acid.

Results

Expression and purification of recombinant ID1-ID2 segment of VAR2CSA

Induced T2053 ID2-ID2a mice antiserum was used in a competition ELISA to examine the antigenic diversity of ID1-ID2a and investigate the level of strain-specificity of induced antibody to ID1-ID2a variants. Competition ELISA was required as it allows to better capture the level of cross-reactivity and allele-specificity of antibodies as compared to standard ELISA. A very low inhibition was observed in the ability of recombinant ID1-ID2a from FCR3 and 3D7 to interfere with the recognition of the homologous T2053 recombinant protein by specific mice antisera. Cross-reactivity of antibodies to FCR3 and 3D7 allele was rather low, with 28.5% and 19.2% respectively cross-reactivity observed (Figure 4).

Naturally acquired antibody recognition profile against ID1-ID2a variants of FCR3 and T2053

A parity-dependent recognition profile of IgG against the FCR3 and T2053 recombinant proteins was seen among sera samples collected at delivery from pregnant women of Ghana. However, a comparative study between the two recombinant proteins clearly shows that women's plasma contained more antibodies against the FCR3 protein than T2053 as a whole. When the women were separated according to their parity status, the same differences were observed with greater recognition of the FCR3 antigen (P < 0.0001). Conversely, IgG recognition among primigravid women of FCR3 ID1-ID2a was similar to the capacity of multigravid women to recognize the recombinant T2053 ID1ID2a (P = 0.99).

Discussion

Currently, VAR2CSA is recognized as undoubtedly the ligand mediating IE adhesion to CSA and, therefore, as the leading vaccine candidate aiming to protect pregnant women living in malaria-endemic areas against the adverse effects of PM. Although several progress have been made in developing subunit vaccines that induce crossreactive antibodies, structural polymorphism in VAR2CSA expressed by field isolates represents a bottleneck in developing a successful vaccine that could stand different parasite strains. Recent genomic analysis of the N-terminal segment of VAR2CSA identified a distinct variant that infects multigravid women already possessing high anti-VAR2CSA IgG. The intriguing feature of these infections with these variants among multigravidae was that they were associated with high parasite density (11). In this study, we investigated the antigenic and binding properties of the ID1-ID2a construct of an African isolate of this type (T2053) in comparison with those of the FCR3 and 3D7 variants which are more common. We were able to successfully express the ID1-ID2a variants of VAR2CSA from T2053, FCR3, and 3D7 strains in an appropriate E. coli Shuffle strain and showed that these proteins can bind to CSA although at different affinities. We further showed that T2053 recombinant ID1-ID2a was immunogenic and could induce antibodies in small animal models. However, antibodies induced against T2053 ID1-ID2a could only weakly recognize similar recombinant ID1-ID2a protein constructs or native VAR2CSA from either FCR3 or 3D7 parasite strains.

This observation clearly suggests that the ID1-ID2a constructs of T2053 express epitopes that are very distinct from those exhibited by the FCR3 and 3D7 variants, although these constructs retain a functional property of binding to CSA. Therefore, it is questionable how this binding function can selectively escape the inhibitory capacities of the acquired antibodies. When analyzing the recognition of the recombinant proteins by the plasmas of pregnant women naturally exposed to P. falciparum, naturally acquired