A Genotyping Study in Benin Comparing the Carriage of Plasmodium falciparum Infections Before Pregnancy and in Early Pregnancy: Story of a Persistent Infection.

BACKGROUND: Malaria infections in the first trimester of pregnancy are frequent and deleterious for both mother and child health. To investigate if these early infections are newly acquired or already present in the host, we assessed whether parasites detected before pregnancy and those detected in early pregnancy are the same infection. METHODS: We used data from the preconceptional "RECIPAL" study (Benin, 2014-2017). Sixty-three pregnant women of 411 included who had a malaria infection detected by quantitative polymerase chain reaction both before pregnancy and at the first antenatal care (ANC) visit were selected for this study. Two highly polymorphic markers, msp-2 and glurp, and a fragment-analysis method were used to enumerate the Plasmodium falciparum genotypes and to quantify their proportions within isolates. An infection was considered as persistent when identical msp-2 and glurp genotypes were found in the corresponding prepregnancy and early-pregnancy samples. RESULTS: The median time between the 2 malaria screenings was 3 months. The median gestational age at the first ANC visit was 6.4 weeks. Most infections before pregnancy were submicroscopic infections. Based on both msp-2 and glurp genotyping, the infection was similar before and in early pregnancy in 46% (29/63) of cases. CONCLUSIONS: Almost half of P. falciparum infections detected in the first trimester originate before pregnancy. Protecting young women from malaria infection before pregnancy might reduce the prevalence of malaria in early pregnancy and its related poor maternal and birth outcomes.

Effect of Drug Pressure on Promoting the Emergence of Antimalarial-Resistant Parasites among Pregnant Women in Ghana.

The continuous spread of antimalarial drug resistance is a threat to current chemotherapy efficacy. Therefore, characterizing the genetic diversity of drug resistance markers is needed to follow treatment effectiveness and further update control strategies. Here, we genotyped Plasmodium falciparum resistance gene markers associated with sulfadoxine-pyrimethamine (SP) and artemisinin-based combination therapy (ACT) in isolates from pregnant women in Ghana. The prevalence of the septuple IRN I- A/FG K GS/Tpfdhfr/pfdhps haplotypes, including the pfdhps A581G and A613S/T mutations, was high at delivery among post-SP treatment isolates (18.2%) compared to those of first antenatal care (before initiation of intermittent preventive treatment of malaria in pregnancy with sulfadoxine-pyrimethamine [IPTp-SP]; 6.1%; P = 0.03). Regarding the pfk13 marker gene, two nonsynonymous mutations (N458D and A481C) were detected at positions previously related to artemisinin resistance in isolates from Southeast Asia. These mutations were predicted in silico to alter the stability of the pfk13 propeller-encoding domain. Overall, these findings highlight the need for intensified monitoring and surveillance of additional mutations associated with increased SP resistance as well as emergence of resistance against artemisinin derivatives.

Correction to: Prevalence and clinical impact of malaria infections detected with a highly sensitive HRP2 rapid diagnostic test in Beninese pregnant women.

An amendment to this paper has been published and can be accessed via the original article.

Clinical Outcomes of Submicroscopic Infections and Correlates of Protection of VAR2CSA Antibodies in a Longitudinal Study of Pregnant Women in Colombia.

Malaria in pregnancy can cause serious adverse outcomes for the mother and the fetus. However, little is known about the effects of submicroscopic infections (SMIs) in pregnancy, particularly in areas where Plasmodium falciparum and Plasmodium vivax cocirculate. A cohort of 187 pregnant women living in Puerto Libertador in northwest Colombia was followed longitudinally from recruitment to delivery. Malaria was diagnosed by microscopy, reverse transcription-quantitative PCR (RT-qPCR), and placental histopathology. Gestational age, hemoglobin concentration, VAR2CSA-specific IgG levels, and adhesion-blocking antibodies were measured during pregnancy. Statistical analyses were performed to evaluate the impact of SMIs on birth weight and other delivery outcomes. Twenty-five percent of women (45/180) were positive for SMIs during pregnancy. Forty-seven percent of infections (21/45) were caused by P. falciparum, 33% were caused by P. vivax, and 20% were caused by mixed Plasmodium spp. Mixed infections of P. falciparum and P. vivax were associated with lower gestational age at delivery (P = 0.0033), while other outcomes were normal. Over 60% of women had antibodies to VAR2CSA, and there was no difference in antibody levels between those with and without SMIs. The anti-adhesion function of these antibodies was associated with protection from SMI-related anemia at delivery (P = 0.0086). SMIs occur frequently during pregnancy, and while mixed infections of both P. falciparum and P. vivax were not associated with a decrease in birth weight, they were associated with significant risk of preterm birth. We propose that the lack of adverse delivery outcomes is due to functional VAR2CSA antibodies that can protect pregnant women from SMI-related anemia.

Persistent Plasmodium falciparum Infection in Women With an Intent to Become Pregnant as a Risk Factor for Pregnancy-associated Malaria.

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.

Parasites Causing Cerebral Falciparum Malaria Bind Multiple Endothelial Receptors and Express EPCR and ICAM-1-Binding PfEMP1.

Background: Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) mediates the binding and accumulation of infected erythrocytes (IE) to blood vessels and tissues. Specific interactions have been described between PfEMP1 and human endothelial proteins CD36, intercellular adhesion molecule-1 (ICAM-1), and endothelial protein C receptor (EPCR); however, cytoadhesion patterns typical for pediatric malaria syndromes and the associated PfEMP1 members are still undefined. Methods: In a cohort of 94 hospitalized children with malaria, we characterized the binding properties of IE collected on admission, and var gene transcription using quantitative polymerase chain reaction. Results: IE from patients with cerebral malaria were more likely to bind EPCR and ICAM-1 than IE from children with uncomplicated malaria (P = .007). The level of transcripts encoding CIDRα1.4 and CIDRα1.5 domain subclasses was higher in patients with severe disease (P < .05). IE populations exhibiting binding to all 3 receptors had higher levels of transcripts encoding PfEMP1 with CIDRα1.4 and Duffy binding-like (DBL)-ß3 domains than parasites, which only bound CD36. Conclusions: These results underpin the significance of EPCR binding in pediatric malaria patients that require hospital admission, and support the notion that complementary receptor interactions of EPCR binding PfEMP1with ICAM-1 amplifies development of severe malaria symptoms.

High plasma levels of soluble endothelial protein C receptor are associated with increased mortality among children with cerebral malaria in Benin.

Loss of endothelial protein C receptor (EPCR) occurs at the sites of Plasmodium falciparum-infected erythrocyte sequestration in patients with or who died from cerebral malaria. In children presenting with different clinical syndromes of malaria, we assessed the relationships between endogenous plasma soluble EPCR (sEPCR) levels and clinical presentation or mortality. After adjustment for age, for treatment before admission, and for a known genetic factor, sEPCR level at admission was positively associated with cerebral malaria (P = .011) and with malaria-related mortality (P = .0003). Measuring sEPCR levels at admission could provide an early biological marker of the outcome of cerebral malaria.

Submicroscopic Plasmodium falciparum Infections Are Associated With Maternal Anemia, Premature Births, and Low Birth Weight.

BACKGROUND: Molecular, as opposed to microscopic, detection measures the real prevalence of Plasmodium falciparum infections. Such occult infections are common during pregnancy but their impact on pregnancy outcomes is unclear. We performed a longitudinal study to describe that impact. METHODS: In a cohort of 1037 Beninese pregnant women, we used ultrasound to accurately estimate gestational ages. Infection with P. falciparum, hemoglobin concentration, use of intermittent preventive treatment during pregnancy (IPTp) for malaria, and other parameters were recorded during pregnancy. Using multivariate analyses, we evaluated the impact of submicroscopic infections on maternal anemia, premature birth, and low birth weight. RESULTS: At inclusion, polymerase chain reaction (PCR) and microscopy detected infection in 40% and 16% of women, respectively. The proportion infected declined markedly after 2 doses of IPTp but rebounded to 34% (by PCR) at delivery. Submicroscopic infections during pregnancy were associated with lower mean hemoglobin irrespective of gravidity, and with increased anemia risk in primigravidae (odds ratio [OR], 2.23; 95% confidence interval [CI], .98-5.07). Prospectively, submicroscopic infections at inclusion were associated with significantly increased risks of low birth weight in primigravidae (OR, 6.09; 95% CI, 1.16-31.95) and premature births in multigravidae (OR, 2.25; 95% CI, 1.13-4.46). CONCLUSIONS: In this detailed longitudinal study, we document the deleterious impact of submicroscopic P. falciparum parasitemia during pregnancy on multiple pregnancy outcomes. Parasitemia occurs frequently during pregnancy, but routine microscopic and rapid diagnostic tests fail to detect the vast majority of episodes. Our findings imply caution in any revision of the current strategies for prevention of pregnancy-associated malaria.

Beninese children with cerebral malaria do not develop humoral immunity against the IT4-VAR19-DC8 PfEMP1 variant linked to EPCR and brain endothelial binding.

BACKGROUND: Malaria is still one of the most prevalent infectious diseases in the world. Sequestration of infected erythrocytes (IEs) is the prime mediator of disease. Cytoadhesion of IEs is mediated by members of the highly diverse Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1). A restricted sub-set of var genes encoding for PfEMP1s possessing the domain cassettes DC8 and DC13 were found to bind to the endothelial protein C receptor (EPCR). These var genes were shown to be highly expressed by parasites from patients with severe malaria clinical outcomes compared to those from patients with uncomplicated symptoms. METHODS: In order to further study the molecular mechanisms underlying DC8/DC13 expressing IEs adhesion to EPCR, a method was developed to produce highly pure recombinant EPCR. The IT4 parasite strain was selected on either anti-IT4-VAR19 purified IgG, EPCR or human brain endothelial cell line and their var gene expression profiles as well as their binding phenotypes were compared. The N-terminal region of IT4-VAR19 comprising a full-length DC8 cassette as well as the single EPCR binding CIDRα1.1 domain were also produced, and their immune recognition (IgG) was assessed using plasma samples from Beninese children presenting acute mild malaria, severe malaria or cerebral malaria at the time of their admission to the clinic, and from convalescent-phase plasma collected 30 days after anti-malarial treatment. RESULTS: The multi-domain VAR19-NTS-DBLγ6 binds to EPCR with a greater affinity than the CIDRα1.1 domain alone and this study also demonstrates that VAR19-NTS-DBLγ6 binding to the EPCR-expressing endothelial cell line (HBEC5i) is more pronounced than that of the CIDRα1.1 domain alone. IT4-VAR19 represents the preferentially expressed-PfEMP1 when FCR3-IEs are selected based on their capability to bind EPCR. Notably, no significant difference in the levels of antibodies towards IT4-VAR19 antigens was observed within all clinical groups between plasma samples collected during the acute malaria phase compared to samples collected 30 days after anti-malaria treatment. CONCLUSIONS: These data indicate that even being the preferentially selected IT4-EPCR-binding variant, the IT4-VAR19-DC8 region does not appear to be associated with the acquisition of antibodies during a single severe paediatric malaria episode in Benin.

First-trimester Plasmodium falciparum infections display a typical "placental" phenotype.

BACKGROUND: Plasmodium falciparum-infected erythrocytes (IEs) adhere to host cell receptors, allowing parasites to sequester into deep vascular beds of various organs. This defining phenomenon of malaria pathogenesis is key to the severe clinical complications associated with cerebral and placental malaria. The principal ligand associated with the binding to chondroitin sulfate A (CSA) that allows placental sequestration of IEs is a P. falciparum erythrocyte membrane protein 1 (PfEMP1) family member encoded by the var2csa gene. METHODS: Here, we investigated the transcription pattern of var genes by real-time polymerase chain reaction, the expression of VAR2CSA, protein by flow cytometry, and the CSA-binding ability of IEs collected at different stages of pregnancy using a static-based Petri dish assay. RESULTS: Through comparison with the profiles of isolates from nonpregnant hosts, we report several lines of evidence showing that parasites infecting women during pregnancy preferentially express VAR2CSA protein, and that selection for the capacity to adhere to CSA via VAR2CSA expression occurs early in pregnancy. CONCLUSIONS: Our data suggest that the placental tropism of P. falciparum is already established in the first trimester of pregnancy, with consequent implications for the development of the pathology associated with placental malaria.

Effectiveness of Intermittent Preventive Treatment with Sulfadoxine-Pyrimethamine in Pregnancy: Low Coverage and High Prevalence of Plasmodium falciparum dhfr-dhps Quintuple Mutants as Major Challenges in Douala, an Urban Setting in Cameroon.

Intermittent preventive treatment in pregnancy with sulfadoxine and pyrimethamine (IPTp-SP) is a key component in the malaria control strategy implemented in Africa. The aim of this study was to determine IPTp-SP adherence and coverage, and the impact on maternal infection and birth outcomes in the context of widespread SP resistance in the city of Douala, Cameroon. Clinical and demographic information were documented among 888 pregnant women attending 3 health facilities, from the antenatal care visit to delivery. Positive samples were genotyped for P. falciparum gene (dhfr, dhps, and k13) mutations. The overall IPTp-SP coverage (≥three doses) was 17.5%, and 5.1% received no dose. P. falciparum prevalence was 16%, with a predominance of submicroscopic infections (89.3%). Malaria infection was significantly associated with locality and history of malaria, and it was reduced among women using indoor residual spraying. Optimal doses of IPTp-SP were significantly associated with reduced infection among newborns and women (secundiparous and multiparous), but there was no impact of IPTp-SP on the newborn bodyweight. Pfdhfr-Pfdhps quintuple mutants were over-represented (IRNI-FGKAA, IRNI-AGKAA), and sextuple mutants (IRNI-AGKAS, IRNI-FGEAA, IRNI-AGKGS) were also reported. The Pfk13 gene mutations associated with artemisinin resistance were not detected. This study highlights the role of ANC in achieving optimal SP coverage in pregnant women, the mitigated impact of IPTp-SP on malaria outcomes, and the high prevalence of multiple SP-resistant P. falciparum parasites in the city of Douala that could compromise the efficacy of IPTp-SP.

The NTS-DBL2X region of VAR2CSA induces cross-reactive antibodies that inhibit adhesion of several Plasmodium falciparum isolates to chondroitin sulfate A.

BACKGROUND: Binding to chondroitin sulfate A by VAR2CSA, a parasite protein expressed on infected erythrocytes, allows placental sequestration of Plasmodium falciparum-infected erythrocytes. This leads to severe consequences such as maternal anemia, stillbirths, and intrauterine growth retardation. The latter has been clearly associated to increased morbidity and mortality of the infants. Acquired anti-VAR2CSA antibodies have been associated with improved pregnancy outcomes, suggesting a vaccine could prevent the syndrome. However, identifying functionally important regions in the large VAR2CSA protein is difficult. METHODS: Using genetic immunization, we raised polyclonal antisera against overlapping segments of VAR2CSA in mice and rabbits. The adhesion-inhibition capacities of induced antisera and of specific antibodies purified from plasma of malaria-exposed pregnant women were assessed on laboratory-adapted parasite lines and field isolates expressing VAR2CSA. Competition enzyme-linked immunosorbent assay (ELISA) was employed to analyze functional resemblance between antibodies induced in animals and those naturally acquired by immune multigravidae. RESULTS: Antibodies targeting the N-terminal sequence (NTS) up to DBL2X (NTS-DBL2X) efficiently blocked parasite adhesion to chondroitin sulfate A in a manner similar to that of antibodies raised against the entire VAR2CSA extracellular domain. Interestingly, naturally acquired antibodies and those induced by vaccination against NTS-DBL2X target overlapping strain-transcendent anti-adhesion epitopes. CONCLUSIONS: This study highlights an important step achieved toward development of a protective vaccine against placental malaria.

Progress and new horizons toward a VAR2CSA-based placental malaria vaccine.

Introduction: Several malaria vaccines are under various phases of development with some promising results. In placental malaria (PM) a deliberately anti-disease approach is considered as many studies have underlined the key role of VAR2CSA protein, which therefore represents the leading vaccine candidate. However, evidence indicates that VAR2CSA antigenic polymorphism remains an obstacle to overcome.Areas covered: This review analyzes the progress made thus far in developing a VAR2CSA-based vaccine, and addresses the current issues and challenges that must be overcome to develop an effective PM vaccine.Expert opinion: Phase I trials of PAMVAC and PRIMVAC VAR2CSA vaccines have shown more or less satisfactory results with regards to safety and immunogenicity. The second generation of VAR2CSA-based vaccines could benefit from optimization approaches to broaden the activity spectrum against various placenta-binding isolates through continued advances in the structural understanding of the interaction with CSA.

Progress and Insights Toward an Effective Placental Malaria Vaccine.

In areas where Plasmodium falciparum transmission is endemic, clinical immunity against malaria is progressively acquired during childhood and adults are usually protected against the severe clinical consequences of the disease. Nevertheless, pregnant women, notably during their first pregnancies, are susceptible to placental malaria and the associated serious clinical outcomes. Placental malaria is characterized by the massive accumulation of P. falciparum infected erythrocytes and monocytes in the placental intervillous spaces leading to maternal anaemia, hypertension, stillbirth and low birth weight due to premature delivery, and foetal growth retardation. Remarkably, the prevalence of placental malaria sharply decreases with successive pregnancies. This protection is associated with the development of antibodies directed towards the surface of P. falciparum-infected erythrocytes from placental origin. Placental sequestration is mediated by the interaction between VAR2CSA, a member of the P. falciparum erythrocyte membrane protein 1 family expressed on the infected erythrocytes surface, and the placental receptor chondroitin sulfate A. VAR2CSA stands today as the leading candidate for a placental malaria vaccine. We recently reported the safety and immunogenicity of two VAR2CSA-derived placental malaria vaccines (PRIMVAC and PAMVAC), spanning the chondroitin sulfate A-binding region of VAR2CSA, in both malaria-naïve and P. falciparum-exposed non-pregnant women in two distinct Phase I clinical trials (ClinicalTrials.gov, NCT02658253 and NCT02647489). This review discusses recent advances in placental malaria vaccine development, with a focus on the recent clinical data, and discusses the next clinical steps to undertake in order to better comprehend vaccine-induced immunity and accelerate vaccine development.

Plasmodium falciparum VAR2CSA-Specific IgG Subclass Responses Reflect Protection Against Low Birth Weight and Pregnancy-Associated Malaria.

Sequestration of Plasmodium falciparum-infected erythrocytes expressing the VAR2CSA antigen in the placenta results in poor pregnancy outcomes, including low birth weight and maternal anemia. Antigen-specific antibody-mediated immunity is acquired during successive pregnancies. Thus, evaluating VAR2CSA-specific IgG profiles among pregnant women will increase knowledge on the immunological mechanisms associated with protection, and help in the development of VAR2CSA-based placental malaria vaccines. Using the PAMVAC candidate vaccine antigen, we assessed anti-VAR2CSA IgG subclass responses of a cohort of pregnant Beninese, and analyzed their relationships with pregnancy outcomes. Cytophilic IgG1 and IgG3 responses were the most frequent, with prevalences ranging from 28% (IgG3) up to 50% (IgG1). Elevated levels of VAR2CSA-specific total IgG and cytophilic IgG3 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.

Afucosylated Plasmodium falciparum-specific IgG is induced by infection but not by subunit vaccination.

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family members mediate receptor- and tissue-specific sequestration of infected erythrocytes (IEs) in malaria. Antibody responses are a central component of naturally acquired malaria immunity. PfEMP1-specific IgG likely protects by inhibiting IE sequestration and through IgG-Fc Receptor (FcγR) mediated phagocytosis and killing of antibody-opsonized IEs. The affinity of afucosylated IgG to FcγRIIIa is up to 40-fold higher than fucosylated IgG, resulting in enhanced antibody-dependent cellular cytotoxicity. Most IgG in plasma is fully fucosylated, but afucosylated IgG is elicited in response to enveloped viruses and to paternal alloantigens during pregnancy. Here we show that naturally acquired PfEMP1-specific IgG is strongly afucosylated in a stable and exposure-dependent manner, and efficiently induces FcγRIIIa-dependent natural killer (NK) cell degranulation. In contrast, immunization with a subunit PfEMP1 (VAR2CSA) vaccine results in fully fucosylated specific IgG. These results have implications for understanding protective natural- and vaccine-induced immunity to malaria.

Plasmodium falciparum parasites causing cerebral malaria share variant surface antigens, but are they specific?

BACKGROUND: Variant surface antigens (VSA) expressed on the surface of Plasmodium falciparum-infected red blood cells constitute a key for parasite sequestration and immune evasion. In distinct malaria pathologies, such as placental malaria, specific antibody response against VSA provides protection. This study investigated the antibody response specifically directed against VSA expressed by parasites isolated from individuals presenting a given type of clinical presentation. METHODS: Plasma and isolates were obtained from four groups of Beninese subjects: healthy adults, patients presenting uncomplicated malaria (UM), cerebral malaria (CM), or pregnancy-associated malaria (PAM). The reactivity of plasma samples from each clinical group was measured by flow cytometry against parasites isolated from individuals from each clinical group. RESULTS: Antibody responses against VSAUM were predominant in CM, UM and HA plasmas. When analysed according to age in all plasma groups, anti-VSACM and -VSAUM antibody levels were similar until six years of age. In older groups (6-18 and >19 years of age), VSAUM antibody levels were higher than VSACM antibody levels (P = .01, P = .0008, respectively). Mean MFI values, measured in all plasmas groups except the PAM plasmas, remained low for anti-VSAPAM antibodies and did not vary with age. One month after infection the level of anti-VSA antibodies able to recognize heterologous VSACM variants was increased in CM patients. In UM patients, antibody levels directed against heterologous VSAUM were similar, both during the infection and one month later. CONCLUSIONS: In conclusion, this study suggests the existence of serologically distinct VSACM and VSAUM. CM isolates were shown to share common epitopes. Specific antibody response to VSAUM was predominant, suggesting a relative low diversity of VSAUM in the study area.

Differential evolution of anti-VAR2CSA- IgG3 in primigravidae and multigravidae pregnant women infected by Plasmodium falciparum.

BACKGROUND: Pregnant women develop protective anti-VSA IgG1 and IgG3 when infected by Plasmodium falciparum. The major target of IgG from serum of infected pregnant women is VAR2CSA. METHODS: In this study, ELISA was used to compare the level of VAR2CSA DBL5epsilon- specific IgG subclasses at enrolment and at delivery in a cohort of pregnant women in Senegal. All antibody measures were analysed in relation to placental infection according to parity. RESULTS: The results show an interaction between immune response to placental malaria and parity. A higher level of anti- DBL5epsilon- IgG3 at enrolment and a higher increase between enrolment and delivery were found in primigravidae who presented with uninfected placenta at delivery in comparison to those who presented with an infection of the placenta. However, high antibody level at delivery was associated with the infection of the placenta in multigravidae. CONCLUSION: This high level of IgG3 in uninfected primigravidae suggests a protective role of these antibodies in this susceptible group, highlighting the importance of VAR2CSA in general and of some of its variants still to be defined, in the induction of protective immunity to pregnancy malaria.