Profiling the Plasmodium falciparum Erythrocyte Membrane Protein 1-Specific Immununoglobulin G Response Among Ghanaian Children With Hemoglobin S and C.

Members of the Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family are important targets for protective immunity. Abnormal display of PfEMP1 on the surfaces of infected erythrocytes (IEs) and reduced cytoadhesion have been demonstrated in hemoglobin (Hb) AS and HbAC, inherited blood disorders associated with protection against severe P. falciparum malaria. We found that Ghanaian children with HbAS had lower levels of immunoglobulin G against several PfEMP1 variants and that this reactivity increased more slowly with age than in their HbAA counterparts. Moreover, children with HbAS have lower total parasite biomass than those with HbAA at comparable peripheral parasitemias, suggesting impaired cytoadhesion of HbAS IEs in vivo and likely explaining the slower acquisition of PfEMP1-specific immunoglobulin G in this group. In contrast, the function of acquired antibodies was comparable among Hb groups and appears to be intact and sufficient to control parasitemia via opsonization and phagocytosis of IEs.

Cryo-EM reveals the conformational epitope of human monoclonal antibody PAM1.4 broadly reacting with polymorphic malarial protein VAR2CSA.

Malaria during pregnancy is a major global health problem caused by infection with Plasmodium falciparum parasites. Severe effects arise from the accumulation of infected erythrocytes in the placenta. Here, erythrocytes infected by late blood-stage parasites adhere to placental chondroitin sulphate A (CS) via VAR2CSA-type P. falciparum erythrocyte membrane protein 1 (PfEMP1) adhesion proteins. Immunity to placental malaria is acquired through exposure and mediated through antibodies to VAR2CSA. Through evolution, the VAR2CSA proteins have diversified in sequence to escape immune recognition but retained their overall macromolecular structure to maintain CS binding affinity. This structural conservation may also have allowed development of broadly reactive antibodies to VAR2CSA in immune women. Here we show the negative stain and cryo-EM structure of the only known broadly reactive human monoclonal antibody, PAM1.4, in complex with VAR2CSA. The data shows how PAM1.4's broad VAR2CSA reactivity is achieved through interactions with multiple conserved residues of different sub-domains forming conformational epitope distant from the CS binding site on the VAR2CSA core structure. Thus, while PAM1.4 may represent a class of antibodies mediating placental malaria immunity by inducing phagocytosis or NK cell-mediated cytotoxicity, it is likely that broadly CS binding-inhibitory antibodies target other epitopes at the CS binding site. Insights on both types of broadly reactive monoclonal antibodies may aid the development of a vaccine against placental malaria.

Plasmodium vivax and SARS-CoV-2 co-infection in Venezuelan pregnant women: a case series.

BACKGROUND: Malaria-endemic areas are not spared from the impact of coronavirus disease 2019 (COVID-19), leading to co-infection scenarios where overlapping symptoms impose serious diagnostic challenges. Current knowledge on Plasmodium spp. and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) co-infection in pregnant women remains limited, especially in Latin America, where Plasmodium vivax infection is highly prevalent. METHODS: This is a case series of five pregnant women with P. vivax and SARS-CoV-2 co-infection hospitalized in two main malaria referral centers of the Capital District and Bolivar state, Venezuela between March 13, 2020 and December 31, 2021. RESULTS: Clinical and laboratory data from five pregnant women with a mean age of 22 years were analyzed; three of them were in the third trimester of pregnancy. Comorbidities included obesity in two cases, hypertension in one, and asthma in one. Three out of five patients had severe to critical COVID-19 disease. Dry cough, fever, chills, and headache were the most frequent symptoms reported. Laboratory analyses showed elevated aspartate/alanine aminotransferase and creatinine levels, thrombocytopenia, and severe anemia as the most relevant abnormalities. The mean period between symptom onset and a positive molecular test for SARS-CoV-2 infection or positive microscopy for Plasmodium spp. was 4.8 ± 2.5 days and 2.8 ± 1.6 days, respectively. The mean hospital stay was 5.4 ± 7 days. Three women recovered and were discharged from the hospital. Two women died, one from cerebral malaria and one from respiratory failure. Three adverse fetal outcomes were registered, two miscarriages and one stillbirth. CONCLUSION: This study documented a predominance of severe/critical COVID-19 disease and a high proportion of adverse maternal-fetal outcomes among pregnant women with malaria and COVID-19 co-infection. More comprehensive prospective cohort studies are warranted to explore the risk factors, management challenges, and clinical outcomes of pregnant women with this co-infection.

Malaria in pregnancy complications in Southern Venezuela.

BACKGROUND: Pregnant women are particularly vulnerable to malaria infections, increasing the risk of maternal-fetal complications, mainly in high-endemicity areas. However, few studies of malaria in pregnancy (MiP) have been carried out in Latin America, a region with low endemicity and transmission of both, Plasmodium falciparum and Plasmodium vivax. Despite the high malaria burden in Venezuela in the last years, no recent studies of MiP have been conducted. Hence, epidemiological and clinical characteristics of pregnant women with malaria in southern Venezuela are described herein. METHODS: A retrospective study in pregnant women attending at the "Ruíz y Páez" University Hospital Complex, Bolivar state, Venezuela, was carried out between February and October, 2019. Epidemiological, clinical, and laboratory information was analysed. RESULTS: Thirty-seven out of 52 pregnant women analysed were infected with P. vivax. Age ranged between 15 and 39 years, and adolescent pregnancies were common. Malaria infection was diagnosed mainly during the third trimester of pregnancy (63.4%). The distribution of symptoms and signs as well as clinical laboratory values was similar among Plasmodium spp. Although uncomplicated malaria was most frequent, 30% (13/52) had severe anaemia. A high proportion of studied women (44%) presented at least one complication during the pregnancy or delivery. Spontaneous abortion was recorded in four women, and three fetal deaths were observed. Six women had preterm delivery without any further complication. CONCLUSIONS: A high prevalence of maternal-fetal complications was found in the studied population, highlighting the requirement for a careful medical follow up during the prenatal check-ups, which should include routinary malaria tests. Preventive measures as distribution of insecticide-treated mosquito net for pregnant women at risk should also be implemented. Those measures can help to reduce the negative impact of malaria on the newborn and mother.

Binding of human serum proteins to Plasmodium falciparum-infected erythrocytes and its association with malaria clinical presentation.

BACKGROUND: The pathogenesis of Plasmodium falciparum malaria is related to the ability of parasite­infected erythrocytes (IEs) to adhere to the vascular endothelium (cytoadhesion/sequestration) or to surrounding uninfected erythrocytes (rosetting). Both processes are mediated by the expression of members of the clonally variant PfEMP1 parasite protein family on the surface of the IEs. Recent evidence obtained with laboratory-adapted clones indicates that P. falciparum can exploit human serum factors, such as IgM and α2-macroglobulin (α2M), to increase the avidity of PfEMP1-mediated binding to erythrocyte receptors, as well as to evade host PfEMP1-specific immune responses. It has remained unclear whether PfEMP1 variants present in field isolates share these characteristics, and whether they are associated with clinical malaria severity. These issues were investigated here. METHODS: Children 1-12 years reporting with P. falciparum malaria to Hohoe Municipal Hospital, Ghana were enrolled in the study. Parasites from children with uncomplicated (UM) and severe malaria (SM) were collected. Binding of α2M and IgM from non-immune individuals to erythrocytes infected by P. falciparum isolates from 34 children (UM and SM) were analysed by flow cytometry. Rosetting in the presence of IgM or α2M was also evaluated. Experimental results were analysed according to the clinical presentation of the patients. RESULTS: Clinical data from 108 children classified as UM (n = 54) and SM cases (n = 54) were analysed. Prostration, severe malaria anaemia, and hyperparasitaemia were the most frequent complications. Three children were diagnosed with cerebral malaria, and one child died. Parasite isolates from UM (n = 14) and SM (n = 20) children were analysed. Most of the field isolates bound non-immune IgM (33/34), whereas the α2M-binding was less common (23/34). Binding of both non-immune IgM and α2M was higher but not significant in IEs from children with SM than from children with UM. In combination, IgM and α2M supported rosette formation at levels similar to that observed in the presence of 10% human serum. CONCLUSIONS: The results support the hypothesis that binding of non-immune IgM and/or α2M to IEs facilitates rosette formation and perhaps contributes to P. falciparum malaria severity.

IgG Responses to the Plasmodium falciparum Antigen VAR2CSA in Colombia Are Restricted to Pregnancy and Are Not Induced by Exposure to Plasmodium vivax.

Clinical immunity to malaria is associated with the acquisition of IgG specific for members of the Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family of clonally variant antigens on the surface of infected erythrocytes (IEs). The VAR2CSA subtype of PfEMP1 mediates IE binding in the placenta. VAR2CSA-specific IgG is normally acquired only after exposure to placental parasites. However, it was recently reported that men and children from Colombia often have high levels of functional VAR2CSA-specific IgG. This potentially undermines the current understanding of malaria immunity in pregnant women, and we thus conducted a study to assess further the levels of VAR2CSA-specific IgG in pregnant and nonpregnant Colombians. Plasma IgG against two full-length recombinant PfEMP1 proteins (one of the VAR2CSA type and one not) produced in baculovirus-transfected insect cells was detected frequently among Colombian men, children, and pregnant women with acute or previous malaria exposure. In contrast, IgG reactivity to a homologous full-length VAR2CSA-type protein expressed in Chinese hamster ovary (CHO) cells was low and infrequent among the Colombian plasma samples, as was reactivity to both corresponding native PfEMP1 proteins. Moreover, human and rabbit antibodies specific for Plasmodium vivax Duffy-binding protein (PvDBP), a protein with some homology to PfEMP1, did not react with VAR2CSA-type recombinant or native proteins, although the mouse monoclonal and PvDBP-specific antibody 3D10 was weakly reactive with recombinant proteins expressed in baculovirus-transfected insect cells. Our data indicate that the previously reported Colombian IgG reactivity to recombinant VAR2CSA is not malaria specific and that the acquisition of VAR2CSA-specific IgG is restricted to pregnancy, in Colombia and elsewhere.

Malaria in pregnancy: a passive surveillance study of pregnant women in low transmission areas of Colombia, Latin America.

BACKGROUND: Malaria causes a significant burden in highly endemic areas where children and pregnant women are more susceptible to severe disease and death, however, in low transmission settings malaria in pregnant women is less frequent. The aim of this study was to provide information of clinical profile, anti-parasite host immune responses and parasite genotyping of pregnant women with malaria in low endemic areas of Colombia. METHODS: This was a descriptive study conducted through passive surveillance in 1328 individuals from three endemic areas of Córdoba, Nariño and Chocó departments between 2011 and 2013. Trained physicians confirmed the pregnancy status and recorded clinical and epidemiological information. Haematological parameters, as well as hepatic and renal function, anti-malarial antibodies and parasite genotypes were evaluated. RESULTS: A total of 582 women presented with malaria infection, 34 of whom were pregnant (5.8 %), and most were infected by Plasmodium falciparum (n = 24). In 44 % (n = 15) of the women, the infection occurred during the first half of pregnancy. Although uncomplicated disease and parasitaemia ≤20,000 parasites/µL were common (n = 31), three women (8.8 %) infected by P. falciparum were classified as severe cases. Mild to moderate anaemia (68 %) and mild thrombocytopaenia (41 %) were the most frequent blood alterations and in four women acute renal failure was observed. Six women presented a second malaria episode during pregnancy mainly caused by P. vivax (n = 5), although no direct evidence of relapse was found by genotyping. Two out of the six women presenting a second malaria episode had severe malaria. A low prevalence of specific anti-parasite antibodies was found. Microsatellites indicated that all P. vivax infections involved multiple lineages whereas all but one P. falciparum infections harboured single genotypes. CONCLUSIONS: Most malaria infected pregnant women displayed uncomplicated malaria, although a few of them with a second malaria episode presented an increased risk of severe malaria which appeared to be associated with malaria transmission intensity and not with levels of anti-parasite antibodies. The effects of severe malaria in both mother and fetus warrant future studies in low transmission settings.

Clinical and epidemiological aspects of complicated malaria in Colombia, 2007-2013.

BACKGROUND: During the last decade, Colombia presented a significant decrease in malaria clinical cases and associated mortality. However, there is a lack of reliable information about the prevalence and characteristics of complicated malaria cases as well as its association with different Plasmodium species. A description of the epidemiological and clinical aspects of complicated malaria in Colombia is presented here. METHODS: A descriptive study was conducted using data collected between 2007 and 2013 by the Public Health Surveillance System (SIVIGILA). Demographic and clinical features were described. Frequency of complicated malaria cases, annual parasite index (API) and annual percent change (APC) for trend modelling by gender and age were also calculated. RESULTS: A total of 547,542 malaria cases were recorded by SIVIGILA during the study period, of which 2553 (0.47 %) corresponded to complicated cases with similar distribution by Plasmodium vivax and Plasmodium falciparum species. Mixed infections were found in 153 cases (6.0 %). Trend modelling of the API for complicated malaria for all parasite species showed a non-significant increase throughout the years (APC 14.4 %; 95 % CI -4.3 to 36.6 %). Complicated malaria individuals were mostly males (62.2 %) and young adults (median age of 23 years). Notably, 72.4 % of the patients attended for malaria diagnosis >72 h after symptoms onset and 17 % reported malaria episodes in the last 30 days. All patients received anti-malarial treatment, but only 40 % received the first-line as recommended by the Colombian guidelines. Overall, hepatic and renal complications were the most common severe manifestations (63.6 %). Whereas hepatic and pulmonary complications were more common in P. vivax infections, renal and cerebral complications were significantly more frequent in patients with P. falciparum. In contrast with mono-infected patients, severe anaemia and shock were more frequent in patients with mixed infection. CONCLUSION: In contrast with the malaria-decreasing trend over the last years, the complicated malaria trend showed a non-significant annual increase. Therefore, in addition to existing national policies on early diagnosis and prompt anti-malarial treatment, more efforts have to be committed addressing the delayed diagnosis and inadequate treatment found in this study. Improving malaria notification forms, medical assistance skills, and capacity should be prioritized.

Clinical profile of Plasmodium falciparum and Plasmodium vivax infections in low and unstable malaria transmission settings of Colombia.

BACKGROUND: Malaria transmission in Latin America is generally hypoendemic and unstable, with Plasmodium vivax as the most prevalent species. However, only a few studies have been carried out in areas with low and unstable transmission, whereas the clinical profile of malaria has been broadly described in hyperendemic areas. The pattern of clinical manifestations and laboratory findings in low to moderate endemic areas of Colombia is reported here. METHODS: A passive surveillance study was conducted between 2011 and 2013 involving 1,328 patients with Plasmodium falciparum, P. vivax or mixed malaria infections attending malaria points-of-care of four malaria endemic-areas with distinct transmission intensities and parasite distribution. Trained physicians recorded clinical symptoms and signs as well as socio-demographic characteristics of study participants. Haematological, biochemical and urine tests were performed at the time of diagnosis. RESULTS: Out of 1,328 cases, 673 (50.7%) were caused by P. vivax; 650 (48.9%) were due to P. falciparum; and five (0.4%) patients had mixed infections (P. falciparum/P. vivax). Most patients (92.5%) presented with uncomplicated malaria characterized by fever, chills, headache, sweating, myalgia/arthralgia and parasitaemia ≤ 20,000 parasites/µL. Fever, tachycardia, pallor and abdominal pain on palpation were more frequent in P. falciparum patients, whereas mild hepatomegaly and splenomegaly were mostly observed with P. vivax. Non-severe anaemia (Hb 7.0-10.9 g/dL) was observed in 20% of the subjects, whereas severe anaemia (Hb < 7.0 g/dL) was present in four patients. Half of the patients presented thrombocytopaenia regardless of parasite species. Leukopaenia, neutrophilia and monocytosis were frequently observed in patients infected with P. falciparum. Mild-to-moderate biochemical alterations were present in ~25% of the patients, particularly abnormal bilirubin in those with P. falciparum and abnormal transaminases in P. vivax malaria patients. Proteinuria was present in ~50% of the patients regardless of parasite species, whereas haemoglobinuria was more common in P. vivax infections. Only 7.5% of the cases were classified as clinically severe malaria, caused by both P. vivax and P. falciparum. CONCLUSIONS: The high prevalence of uncomplicated malaria associated with moderate parasitaemia suggests the importance of timely diagnosis and effective treatment and encourages new activities to further decrease complicated malaria cases and mortality.

Profiling the antibody response of humans protected by immunization with Plasmodium vivax radiation-attenuated sporozoites.

Malaria sterile immunity has been reproducibly induced by immunization with Plasmodium radiation-attenuated sporozoites (RAS). Analyses of sera from RAS-immunized individuals allowed the identification of P. falciparum antigens, such as the circumsporozoite protein (CSP), the basis for the RTS, S and R21Matrix-M vaccines. Similar advances in P. vivax (Pv) vaccination have been elusive. We previously reported 42% (5/12) of sterile protection in malaria-unexposed, Duffy-positive (Fy +) volunteers immunized with PvRAS followed by a controlled human malaria infection (CHMI). Using a custom protein microarray displaying 515 Pv antigens, we found a significantly higher reactivity to PvCSP and one hypothetical protein (PVX_089630) in volunteers protected against P. vivax infection. In mock-vaccinated Fy + volunteers, a strong antibody response to CHMI was also observed. Although the Fy- volunteers immunized with non-irradiated Pv-infected mosquitoes (live sporozoites) did not develop malaria after CHMI, they recognized a high number of antigens, indicating the temporary presence of asexual parasites in peripheral blood. Together, our findings contribute to the understanding of the antibody response to P. vivax infection and allow the identification of novel parasite antigens as vaccine candidates.Trial registration: ClinicalTrials.gov number: NCT01082341.

Plasmodium falciparum infection and naturally acquired immunity to malaria antigens among Ghanaian children in northern Ghana.

Background: The surge in malaria cases and deaths in recent years, particularly in Africa, despite the widespread implementation of malaria-control measures could be due to inefficiencies in malaria control and prevention measures in malaria-endemic communities. In this context, this study provides the malaria situation report among children in three Municipalities in Northern Ghana, where Seasonal Malaria Chemotherapy (SMC) is implemented by Ghana Health Service (GHS). Methods: A cross-sectional household survey was carried out to assess the malaria knowledge, attitudes, and practices (KAP) and malaria prevalence in 394 households in 13 rural communities in the Kumbugu, Nanton and Tolon Municipalities, Northern Region, Ghana. This was followed by screening for P. falciparum infection with anti-HRP2 RDT and PCR among children 1-17 years in the households. Plasma levels of IgG specific for crude P. falciparum antigen (3D7) and four recombinant malaria antigens (CSP, GLURP, MSP3, and Pfs230) were assessed by ELISA. The malaria and parasitaemia data were converted into frequency and subgroup proportions and disaggregated by study sites and demographic information of the participants. The ELISA data was converted to arbitrary units (AU) and similarly compared across study sites and demographic information. Results: The P. falciparum infection rate and frequency of malaria were high in the study areas with significant age-dependent and inter-community differences, which were reflected by differences in plasma levels of P. falciparum-specific IgG. Over 60% of households reported the use of bed nets and indoor insecticide sprays/coils, and 14% mentioned bush clearing around homes (14%) as malaria preventive measures. Community health centres were the preferred place for households (88%) to seek malaria treatment but over-the-counter drug stores were the major source (66%) of their antimalarials. Overall, malaria preventive and treatment practices were sub-optimal. Conclusions: P. falciparum infection and malaria are still high in the studied communities, indicating that preventive and control measures against the disease in the region remain inadequate. Efforts to ensure high SMC compliance and to improve preventative and treatment practices thus seem cost-beneficial "low-hanging fruits" in the fight against malaria in the Northern Region of Ghana.

Seroprevalence of viral and bacterial pathogens among malaria patients in an endemic area of southern Venezuela.

BACKGROUND: Malaria remains a leading public health problem worldwide. Co-infections with other pathogens complicate its diagnosis and may modify the disease's clinical course and management. Similarities in malaria clinical presentation with other infections and overlapping endemicity result in underdiagnosis of co-infections and increased mortality. Thus, the aim of this study was to determine the seroprevalence of viral and bacterial pathogens among diagnosed malaria patients in malaria-endemic areas in Venezuela. METHODS: A cross-sectional study was conducted on malaria patients attending three reference medical centres in Ciudad Bolivar, Venezuela. Clinical evaluation and laboratory tests for dengue virus (DENV), chikungunya virus (CHIKV), viral hepatitis [hepatitis A virus (HAV), hepatitis B virus (HBV), and hepatitis C virus (HCV)], and leptospirosis (LEP) were performed by enzyme-linked immunosorbent assays. Previous exposure to these pathogens was defined by the presence of specific immunoglobulin (Ig) G, and co-infection or recent exposure (CoRE) was determined by the presence of specific IgM alone or IgM + IgG. Data analysis considered descriptive statistics. Parameter distribution was statistically evaluated using Kolmogorov-Smirnov test and the necessary comparison tests. Odds ratio (OR) for complications was determined according to CoRE presence with a 95% confidence interval (CI). RESULTS: A total of 161 malaria patients were studied, 66% infected with Plasmodium vivax, 27% with P. falciparum, and 7.5% harboured P. vivax/P. falciparum mixed infection. Previous exposure to DENV (60%) and CHIKV (25%) was frequent. CoRE was confirmed in 55 of the 161 malaria patients (34%) and were more frequent in P. falciparum (49%) than in P. vivax (29%) and mixed malaria patients (25%) (OR = 2.43, 95% CI: 1.39-4.25, P = 0.018). The most frequent CoRE was DENV (15%), followed by HAV (12%), HBV (6.2%), CHIKV (5.5%), and LEP (3.7%); HCV CoRE was absent. Complicated malaria was significantly more frequent in patients with CoRE (56%) than those without CoRE (36%; OR = 2.31, 95% CI: 1.18-4.92, P = 0.013). CONCLUSIONS: We found high CoRE prevalence in malaria patients as determined by serology in the study region; cases were associated with a worse clinical outcome. Further prospective studies with samples from different infection sites and the use of molecular tools are needed to determine the clinical significance of these findings.

Anti-Plasmodium falciparum invasion ligand antibodies in a low malaria transmission region, Loreto, Peru.

BACKGROUND: Erythrocyte invasion by Plasmodium falciparum is a complex process that involves two families; Erythrocyte Binding-Like (EBL) and the Reticulocyte Binding-Like (PfRh) proteins. Antibodies that inhibit merozoite attachment and invasion are believed to be important in mediating naturally acquired immunity and immunity generated by parasite blood stage vaccine candidates. The hypotheses tested in this study were 1) that antibody responses against specific P. falciparum invasion ligands (EBL and PfRh) differ between symptomatic and asymptomatic individuals living in the low-transmission region of the Peruvian Amazon and 2), such antibody responses might have an association, either direct or indirect, with clinical immunity observed in asymptomatically parasitaemic individuals. METHODS: ELISA was used to assess antibody responses (IgG, IgG1 and IgG3) against recombinant P. falciparum invasion ligands of the EBL (EBA-175, EBA-181, EBA-140) and PfRh families (PfRh1, PfRh2a, PfRh2b, PfRh4 and PfRh5) in 45 individuals infected with P. falciparum from Peruvian Amazon. Individuals were classified as having symptomatic malaria (N=37) or asymptomatic infection (N=8). RESULTS: Antibody responses against both EBL and PfRh family proteins were significantly higher in asymptomatic compared to symptomatic individuals, demonstrating an association with clinical immunity. Significant differences in the total IgG responses were observed with EBA-175, EBA-181, PfRh2b, and MSP119 (as a control). IgG1 responses against EBA-181, PfRh2a and PfRh2b were significantly higher in the asymptomatic individuals. Total IgG antibody responses against PfRh1, PfRh2a, PfRh2b, PfRh5, EBA-175, EBA-181 and MSP119 proteins were negatively correlated with level of parasitaemia. IgG1 responses against EBA-181, PfRh2a and PfRh2b and IgG3 response for PfRh2a were also negatively correlated with parasitaemia. CONCLUSIONS: These data suggest that falciparum malaria patients who develop clinical immunity (asymptomatic parasitaemia) in a low transmission setting such as the Peruvian Amazon have antibody responses to defined P. falciparum invasion ligand proteins higher than those found in symptomatic (non-immune) patients. While these findings will have to be confirmed by larger studies, these results are consistent with a potential role for one or more of these invasion ligands as a component of an anti-P. falciparum vaccine in low-transmission malaria-endemic regions.

Single-Cell Sorting of Plasmodium falciparum-Infected Erythrocytes Expressing Particular PfEMP1 Variants.

Cultures of Plasmodium falciparum often contain a heterogeneous parasite population. However, several studies require analysis of single infected erythrocytes (IEs) or a clonal parasite population derived from a single parasite. This protocol describes an efficient method for cloning by using fluorescence-activated cell sorting (FACS). For this, an antibody for a particular IEs surface protein it is added to the cell mixture to separate positive and negative IEs for that marker. After the separation, the viable homogeneous population can be used to grow in culture or for molecular analysis.

Establishing and Maintaining In Vitro Cultures of Asexual Blood Stages of Plasmodium falciparum.

In vitro culture of asexual blood stages of Plasmodium falciparum is essential to study the parasite biology, and several aspects need to be addressed to successfully cultivate the parasites, including the requirements for red blood cells and specific nutrients. Since Trager and Jensen established the technique in 1976, some modifications have been introduced to improve the growth rate and yield. In brief, the method is based on the use of human red blood cells suspended in RPMI-1640 culture medium supplemented with a source of lipids and maintained at 37 °C in a low-oxygen atmosphere. In this protocol, a description of thawing, culturing, and cryopreservation of asexual blood stages of P. falciparum is presented.

Analysis by Flow Cytometry of α2-Macroglobulin and Nonimmune IgM-Binding to Plasmodium falciparum-Infected Erythrocytes.

Several members of the Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family can bind human serum proteins such as IgM and α2-macroglobulin (α2M). This binding seems to play a role in pathogenesis and immune evasion by improving the avidity of PfEMP1-mediated binding to erythrocyte receptors and/or by masking antibody epitopes in PfEMP1. In this protocol, we describe a flow cytometry-based protocol to evaluate IgM- and α2M-binding to intact and unfixed mature-stage IEs. The method can be used for laboratory clones and field isolates.

Immunomagnetic Selection of Plasmodium falciparum-Infected Erythrocytes Expressing Particular PfEMP1 Variants.

Plasmodium falciparum expresses a broad range of proteins on the surface of infected erythrocytes (IEs), including members of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family. This protocol describes an immunomagnetic selection method using PfEMP1-specific antibodies to obtain a parasite clone homogenously expressing a particular PfEMP1 protein. The expression of the corresponding PfEMP1 is later tested by flow cytometry, and the selected parasites can be used for further analysis.

No sweet deal: the antibody-mediated immune response to malaria.

IgG antibodies are key effector molecules in acquired immunity to Plasmodium falciparum malaria, and the PfEMP1 adhesins expressed on the surface of the infected erythrocytes are crucial immunological targets. The antigen specificity of these antibodies has therefore been a major research focus. However, we recently reported that the Fc domain of naturally induced PfEMP1-specific IgG1 is selectively modified by post-translational omission of fucose from the conserved Fc glycan. The resulting afucosylated IgG has increased affinity for the IgG-Fc-receptor III family (FcγRIII), found on natural killer cells and on subsets of other cells in the immune system. We discuss the implications of these findings for the basic understanding of antimalarial immunity and for the design of improved vaccines against the disease.

Natural Acquired Immunity to Malaria Antigens among Pregnant Women with Hemoglobin C Trait.

Hemoglobin C is the second most common structural hemoglobinopathy in Africa, and carriers have a reduced risk of severe malaria. However, the effect of HbAC on the antibody response to malaria antigens in pregnancy has not been studied. Here, we measured PfEMP1-specific antibodies in plasma samples from 74 Beninese pregnant women with either HbAA or HbAC. IgG-mediated inhibition of VAR2CSA+ infected erythrocytes adhesion to chondroitin sulfate A (CSA) was also tested. PfEMP1-specific IgG levels to VAR2CSA were significantly lower in HbAC women, suggesting less exposure to VAR2CSA. In contrast, the percentage of VAR2CSA+-infected erythrocytes adhesion to CSA was not different between HbAA and HbAC women. Moreover, IgG levels to PfEMP1 variants associated with severe malaria were not significantly different between groups. The findings indicate similar exposure to Plasmodium falciparum parasites expressing PfEMP1 variants causing severe malaria, and justify more comprehensive studies of hemoglobinopathy-related qualitative and quantitative differences in PfEMP1-specific antibody responses.

PfEMP1-Specific Immunoglobulin G Reactivity Among Beninese Pregnant Women With Sickle Cell Trait.

BACKGROUND: Sickle cell trait (HbAS) protects against severe Plasmodium falciparum malaria but not against placental malaria (PM). In this study, P falciparum erythrocyte membrane protein (PfEMP1)-specific antibodies were measured in HbAA and HbAS Beninese pregnant women as a proxy of exposure to specific PfEMP1 variants. METHODS: Plasma samples collected at delivery from 338 HbAA and 63 HbAS women were used to measure immunoglobulin (Ig)G levels to 6 recombinant PfEMP1 proteins and 3 corresponding native proteins expressed on the infected erythrocyte (IE) surface. Immunoglobulin G-mediated inhibition of VAR2CSA+ IEs adhesion to chondroitin sulfate A (CSA) was also tested. RESULTS: Levels of PfEMP1-specific IgG were similar in the 2 groups, except for native IT4VAR09 on IEs, where IgG levels were significantly higher in HbAS women. Adjusted odds ratios for women with positive IgG to HB3VAR06 and PFD1235w suggest a lower risk of infection with these virulent variants among HbAS individuals. The percentage of IEs binding to CSA did not differ between HbAA and HbAS women, but it correlated positively with levels of anti-VAR2CSA and parity. Women with PM had lower levels of anti-VAR2CSA-specific IgG and lower IgG-mediated inhibition of IE adhesion to CSA. CONCLUSIONS: The findings support similar malaria exposure in HbAA and HbAS women and a lack of HbAS-dependent protection against placental infection among pregnant women.