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Abstract Plasmodium falciparum is responsible for malaria-related morbidity and mortality. PfEMP1 (P. falciparum erythrocyte membrane protein 1) mediates infected erythrocytes adhesion to various surface vascular receptors, including intercellular adhesion molecule-1 (ICAM-1), associating this interaction with severe malaria in several studies. Genetic variation in host ICAM-1 plays a significant role in determining susceptibility to malaria infection via clinical phenotypes such as the ICAM-1Kilifi variant which has been reported to be associated with susceptibility in populations. Our genomic and structural analysis of single nucleotide polymorphisms (SNPs) in ICAM-1 revealed 9 unique mutations each in its distinct A-type and BC-type PfEMP1 DBLβ-interacting regions. These mutations are noted in only a few field isolates and mainly in the African/African American population. The ICAM-1Kilifi variant lies in a flexible loop proximal to the DBLβ-interacting region. This analysis will assist in establishing functional correlations of reported global mutations via experimental and clinical studies and in the tailored design of population-specific genetic surveillance studies. Understanding host polymorphism as an evolutionary force in diverse populations can help to predict predisposition to disease severity and will contribute towards laying the framework for designing population-specific personalized medicines for severe malaria.

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. [ABSTRACT FROM AUTHOR]

Plasmodium falciparum is responsible for malaria-related morbidity and mortality. PfEMP1 (P. falciparum erythrocyte membrane protein 1) mediates infected erythrocytes adhesion to various surface vascular receptors, including intercellular adhesion molecule-1 (ICAM-1), associating this interaction with severe malaria in several studies. Genetic variation in host ICAM-1 plays a significant role in determining susceptibility to malaria infection via clinical phenotypes such as the ICAM-1Kilifi variant which has been reported to be associated with susceptibility in populations. Our genomic and structural analysis of single nucleotide polymorphisms (SNPs) in ICAM-1 revealed 9 unique mutations each in its distinct A-type and BC-type PfEMP1 DBLβ-interacting regions. These mutations are noted in only a few field isolates and mainly in the African/African American population. The ICAM-1Kilifi variant lies in a flexible loop proximal to the DBLβ-interacting region. This analysis will assist in establishing functional correlations of reported global mutations via experimental and clinical studies and in the tailored design of population-specific genetic surveillance studies. Understanding host polymorphism as an evolutionary force in diverse populations can help to predict predisposition to disease severity and will contribute towards laying the framework for designing population-specific personalized medicines for severe malaria. [ABSTRACT FROM AUTHOR]

Background Antibodies to variant surface antigens (VSAs) such as Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) may vary with malaria severity. The influence of ABO blood group on antibody development is not understood. Methods Immunoglobulin G antibodies to VSAs in Papua New Guinean children with severe (n = 41) or uncomplicated (n = 30) malaria were measured by flow cytometry using homologous P falciparum isolates. Isolates were incubated with ABO-matched homologous and heterologous acute and convalescent plasma. RNA was used to assess var gene transcription. Results Antibodies to homologous, but not heterologous, isolates were boosted in convalescence. The relationship between antibody and severity varied by blood group. Antibodies to VSAs were similar in severe and uncomplicated malaria at presentation, higher in severe than uncomplicated malaria in convalescence, and higher in children with blood group O than other children. Six var gene transcripts best distinguished severe from uncomplicated malaria, including UpsA and 2 CIDRα1 domains. Conclusions ABO blood group may influence antibody acquisition to VSAs and susceptibility to severe malaria. Children in Papua New Guinea showed little evidence of acquisition of cross-reactive antibodies following malaria. Var gene transcripts in Papua New Guinean children with severe malaria were similar to those reported from Africa. [ABSTRACT FROM AUTHOR]

Reorganization of cell organelle‐deprived host red blood cells by the apicomplexan malaria parasite Plasmodium falciparum enables their cytoadherence to endothelial cells that line the microvasculature. This increases the time red blood cells infected with mature developmental stages remain within selected organs such as the brain to avoid the spleen passage, which can lead to severe complications and cumulate in patient death. The Maurer's clefts are a novel secretory organelle of parasite origin established by the parasite in the cytoplasm of the host red blood cell in order to facilitate the establishment of cytoadherence by conducting the trafficking of immunovariant adhesins to the host cell surface. Another important function of the organelle is the sorting of other proteins the parasite traffics into its host cell. Although the organelle is of high importance for the pathology of malaria, additional putative functions, structure, and genesis remain shrouded in mystery more than a century after its discovery. In this review, we highlight our current knowledge about the Maurer's clefts and other novel secretory organelles established within the host cell cytoplasm by human‐pathogenic malaria parasites and other parasites that reside within human red blood cells. [ABSTRACT FROM AUTHOR]

ABSTRACT Antibody responses to variant surface antigens (VSAs) produced by the malaria parasite Plasmodium falciparum may contribute to age-related natural immunity to severe malaria. One VSA family, P. falciparum erythrocyte membrane protein-1 (PfEMP1), includes a subset of proteins that binds endothelial protein C receptor (EPCR) in human hosts and potentially disrupts the regulation of inflammatory responses, which may lead to the development of severe malaria. We probed peptide microarrays containing segments spanning five PfEMP1 EPCR-binding domain variants with sera from 10 Malian adults and 10 children to determine the differences between adult and pediatric immune responses. We defined serorecognized peptides and amino acid residues as those that elicited a significantly higher antibody response than malaria-naïve controls. We aimed to identify regions consistently serorecognized among adults but not among children across PfEMP1 variants, potentially indicating regions that drive the development of immunity to severe malaria. Adult sera consistently demonstrated broader and more intense serologic responses to constitutive PfEMP1 peptides than pediatric sera, including peptides in EPCR-binding domains. Both adults and children serorecognized a significantly higher proportion of EPCR-binding peptides than peptides that do not directly participate in receptor binding, indicating a preferential development of serologic responses at functional residues. Over the course of a single malaria transmission season, pediatric serological responses increased between the start and the peak of the season, but waned as the transmission season ended. IMPORTANCE Severe malaria and death related to malaria disproportionately affect sub-Saharan children under 5 years of age, commonly manifesting as cerebral malaria and/or severe malarial anemia. In contrast, adults in malaria-endemic regions tend to experience asymptomatic or mild disease. Our findings indicate that natural immunity to malaria targets specific regions within the EPCR-binding domain, particularly peptides containing EPCR-binding residues. Epitopes containing these residues may be promising targets for vaccines or therapeutics directed against severe malaria. Our approach provides insight into the development of natural immunity to a binding target linked to severe malaria by characterizing an “adult-like” response as recognizing a proportion of epitopes within the PfEMP1 protein, particularly regions that mediate EPCR binding. This “adult-like” response likely requires multiple years of malaria exposure, as increases in pediatric serologic response over a single malaria transmission season do not appear significant.

Parasitemia among pregnant women with protective immunity to Plasmodium falciparum malaria is often dominated by VAR2CSA-positive infected erythrocytes (IEs). VAR2CSA mediates sequestration of IEs in the placenta. We hypothesized that the previously observed spontaneous postpartum clearance of parasitemia in such women is related to the expulsion of the placenta, which removes the sequestration focus of VAR2CSA-positive IEs. We assessed parasitemias and gene transcription before and shortly after delivery in 17 Ghanaian women. The precipitous decline in parasitemia postpartum was accompanied by selective reduction in transcription of the gene encoding VAR2CSA. Our findings provide a mechanistic explanation for the earlier observation. [ABSTRACT FROM AUTHOR]

BackgroundPlasmodium falciparum erythrocyte membrane protein-1 (PfEMP1) antigens play a critical role in host immune evasion. Serologic responses to these antigens have been associated with protection from clinical malaria, suggesting that antibodies to PfEMP1 antigens may contribute to natural immunity. The first N-terminal constitutive domain in a PfEMP1 is the Duffy binding-like alpha (DBL-α) domain, which contains a 300 to 400 base pair region unique to each particular protein (the DBL-α "tag"). This DBL-α tag has been used as a marker of PfEMP1 diversity and serologic responses in malaria-exposed populations. In this study, using sera from a malaria-endemic region, responses to DBL-α tags were compared to responses to the corresponding entire DBL-α domain (or "parent" domain) coupled with the succeeding cysteine-rich interdomain region (CIDR).MethodsA protein microarray populated with DBL-α tags, the parent DBL-CIDR head structures, and downstream PfEMP1 protein fragments was probed with sera from Malian children (aged 1 to 6 years) and adults from the control arms of apical membrane antigen 1 (AMA1) vaccine clinical trials before and during a malaria transmission season. Serological responses to the DBL-α tag and the DBL-CIDR head structure were measured and compared in children and adults, and throughout the season.ResultsMalian serologic responses to a PfEMP1's DBL-α tag region did not correlate with seasonal malaria exposure, or with responses to the parent DBL-CIDR head structure in either children or adults. Parent DBL-CIDR head structures were better indicators of malaria exposure.ConclusionsLarger PfEMP1 domains may be better indicators of malaria exposure than short, variable PfEMP1 fragments such as DBL-α tags. PfEMP1 head structures that include conserved sequences appear particularly well suited for study as serologic predictors of malaria exposure.

Acquisition of antibodies to Plasmodium falciparum variant surface antigens (VSA) expressed on infected red blood cells (iRBCs) is associated with naturally acquired immunity to malaria. We have previously shown that antibodies to VSA on iRBCs are associated with protection against parasite growth in the context of controlled human malaria infection (CHMI). This study explored whether antibodies to recombinant antigens derived from Pf EMP1 domains were independently associated with protection during CHMI in semi-immune Kenyan adults. We used a multiplex bead assay to measure levels of IgG antibody against a panel of 27 recombinant Pf EMP1 antigens derived from the Pf EMP1 repertoire of the 3D7 parasite clone. We measured IgG levels in plasma samples collected from the CHMI participants before inoculation with Sanaria® PfSPZ Challenge, on the day of diagnosis, and 35 days post-inoculation. Univariable and multivariable Cox regression analysis was used to evaluate the relationship between the levels of antibodies to the antigens and CHMI outcome. We also adjusted for previous data including antibodies to VSA on iRBCs, and we assessed the kinetics of antibody acquisition to the different Pf EMP1 recombinant antigens over time. All study participants had detectable antibodies to multiple Pf EMP1 proteins before inoculation. All Pf EMP1 antigens were associated with protection against parasite growth to the threshold criteria for treatment in CHMI, albeit with substantial collinearity. However, individual Pf EMP1 antigens were not independently associated with protection following adjustment for breadth of reactivity to VSA on iRBCs and schizont extract. In addition, antibodies to Pf EMP1 antigens derived from group B Pf EMP1 were induced and sustained in the participants who could not control parasite growth. This study shows that the breadth of antibody response to VSA on iRBCs, and not to specific Pf EMP1 antigens, is predictive of protection against malaria in CHMI. • Naturally acquired Antibodies to recombinant Pf EMP1 were linked to protection in Controlled Human Malaria Infection (CHMI). • Antibodies to the Pf EMP1 domains were highly correlated with each other and with antibody breadth to P. falciparum VSAs • Breadth of antibody responses to VSAs on iRBCs is an independent predictor of protection in CHMI. [ABSTRACT FROM AUTHOR]

Abstract Background There is a clear need for novel approaches to malaria vaccine development. We aimed to develop a genetically attenuated blood-stage vaccine and test its safety, infectivity, and immunogenicity in healthy volunteers. Our approach was to target the gene encoding the knob-associated histidine-rich protein (KAHRP), which is responsible for the assembly of knob structures at the infected erythrocyte surface. Knobs are required for correct display of the polymorphic adhesion ligand P. falciparum erythrocyte membrane protein 1 (PfEMP1), a key virulence determinant encoded by a repertoire of var genes. Methods The gene encoding KAHRP was deleted from P. falciparum 3D7 and a master cell bank was produced in accordance with Good Manufacturing Practice. Eight malaria naïve males were intravenously inoculated (day 0) with 1800 (2 subjects), 1.8 × 105 (2 subjects), or 3 × 106 viable parasites (4 subjects). Parasitemia was measured using qPCR; immunogenicity was determined using standard assays. Parasites were rescued into culture for in vitro analyses (genome sequencing, cytoadhesion assays, scanning electron microscopy, var gene expression). Results None of the subjects who were administered with 1800 or 1.8 × 105 parasites developed parasitemia; 3/4 subjects administered 3× 106 parasites developed significant parasitemia, first detected on days 13, 18, and 22. One of these three subjects developed symptoms of malaria simultaneously with influenza B (day 17; 14,022 parasites/mL); one subject developed mild symptoms on day 28 (19,956 parasites/mL); and one subject remained asymptomatic up to day 35 (5046 parasites/mL). Parasitemia rapidly cleared with artemether/lumefantrine. Parasitemia induced a parasite-specific antibody and cell-mediated immune response. Parasites cultured ex vivo exhibited genotypic and phenotypic properties similar to inoculated parasites, although the var gene expression profile changed during growth in vivo. Conclusions This study represents the first clinical investigation of a genetically attenuated blood-stage human malaria vaccine. A P. falciparum 3D7 kahrp– strain was tested in vivo and found to be immunogenic but can lead to patent parasitemia at high doses. Trial registration Australian New Zealand Clinical Trials Registry (number: ACTRN12617000824369 ; date: 06 June 2017).

[Display omitted] • Age-specific Plasmodium falciparum var diversity patterns were measured in a cohort of 1,541 individuals in Ghana. • We define 42,399 distinct var sequence variants organised as non-overlapping repertoires within individuals. • Risk of infection with parasites with diverse var repertoires exists at any age. • High seasonal turnover of parasites with diverse isolate var repertoires was also observed in individuals of all ages. • Simulations illustrate why adolescents and adults remain susceptible to blood stage infection, even with multiple genomes. Immunity to Plasmodium falciparum is non-sterilising, thus individuals residing in malaria-endemic areas are at risk of infection throughout their lifetime. Here we seek to find a genomic epidemiological explanation for why residents of all ages harbour blood stage infections despite lifelong exposure to P. falciparum in areas of high transmission. We do this by exploring, for the first known time, the age-specific patterns of diversity of variant antigen encoding (var) genes in the reservoir of infection. Microscopic and submicroscopic P. falciparum infections were analysed at the end of the wet and dry seasons in 2012–2013 for a cohort of 1541 residents aged from 1 to 91 years in an area characterised by high seasonal malaria transmission in Ghana. By sequencing the near ubiquitous Duffy-binding-like alpha domain (DBLα) that encodes immunogenic domains, we defined var gene diversity in an estimated 1096 genomes detected in sequential wet and dry season sampling of this cohort. Unprecedented var (DBLα) diversity was observed in all ages with 42,399 unique var types detected. There was a high degree of maintenance of types between seasons (>40% seen more than once), with many of the same types, especially upsA, appearing multiple times in isolates from different individuals. Children and adolescents were found to be significant reservoirs of var DBLα diversity compared with adults. Var repertoires within individuals were highly variable, with children having more related var repertoires compared to adolescents and adults. Individuals of all ages harboured multiple genomes with var repertoires unrelated to those infecting other hosts. High turnover of parasites with diverse isolate var repertoires was also observed in all ages. These age-specific patterns are best explained by variant-specific immune selection. The observed level of var diversity for the population was then used to simulate the development of variant-specific immunity to the diverse var types under conservative assumptions. Simulations showed that the extent of observed var diversity with limited repertoire relatedness was sufficient to explain why adolescents and adults in this community remain susceptible to blood stage infection, even with multiple genomes. [ABSTRACT FROM AUTHOR]

Concerted efforts to fight malaria have caused significant reductions in global malaria cases and mortality. Sustaining this will be critical to avoid rebound and outbreaks of seasonal malaria. Identifying predictive attributes that define clinical malaria will be key to guide development of second-generation tools to fight malaria. Broadly reactive antibodies against variable surface antigens that are expressed on the surface of infected erythrocytes and merozoites stage antigens are targets of naturally acquired immunity and prime candidates for anti-malaria therapeutics and vaccines. However, predicting the relationship between the antigen-specific antibodies and protection from clinical malaria remains unresolved. Here, we used new datasets and multiple approaches combined with re-analysis of our previous data to assess the multi-dimensional and complex relationship between antibody responses and clinical malaria outcomes. We observed 22 antigens (17 PfEMP1 domains, 3 RIFIN family members, merozoite surface protein 3 (PF3D7_1035400), and merozoites-associated armadillo repeats protein (PF3D7_1035900) that were selected across three different clinical malaria definitions (1,000/2,500/5,000 parasites/µl plus fever). In addition, Principal Components Analysis (PCA) indicated that the first three components (Dim1, Dim2 and Dim3 with eigenvalues of 306, 48, and 29, respectively) accounted for 66.1% of the total variations seen. Specifically, the Dim1, Dim2 and Dim3 explained 52.8%, 8.2% and 5% of variability, respectively. We further observed a significant relationship between the first component scores and age with antibodies to PfEMP1 domains being the key contributing variables. This is consistent with a recent proposal suggesting that there is an ordered acquisition of antibodies targeting PfEMP1 proteins. Thus, although limited, and further work on the significance of the selected antigens will be required, these approaches may provide insights for identification of drivers of naturally acquired protective immunity as well as guide development of additional tools for malaria elimination and eradication. [ABSTRACT FROM AUTHOR]

Plasmodium falciparum, the deadliest form of human malaria, remains one of the major threats to human health in endemic regions. Its virulence is attributed to its ability to modify infected red blood cells (iRBC) to adhere to endothelial receptors by placing variable antigens known as PfEMP1 on the iRBC surface. PfEMP1 expression determines the cytoadhesive properties of the iRBCs and is implicated in severe malaria. To evade antibody‐mediated responses, the parasite undergoes continuous switches of expression between different PfEMP1 variants. Recently, it became clear that in addition to antibody‐mediated responses, PfEMP1 triggers innate immune responses; however, the role of neutrophils, the most abundant white blood cells in the human circulation, in malaria remains elusive. Here, we show that neutrophils recognize and kill blood‐stage P. falciparum isolates. We identify neutrophil ICAM‐1 and specific PfEMP1 implicated in cerebral malaria as the key molecules involved in this killing. Our data provide mechanistic insight into the interactions between neutrophils and iRBCs and demonstrate the important influence of PfEMP1 on the selective innate response to cerebral malaria. SYNOPSIS: As first line of defense against severe malaria, neutrophils recognize specific PfEMP1 molecules on the infected erythrocyte via ICAM‐1. This ligand‐receptor interaction leads to efficient killing of Plasmodium falciparum parasites associated with cerebral malaria. Neutrophils kill blood stage P. falciparum by phagocytosis of infected erythrocytes and targeted oxidative burst.Neutrophil's ICAM‐1 and parasite's PfEMP1 are important mediators of neutrophil interactions with infected erythrocytes.Neutrophils impose strong selective pressure against parasites expressing PfEMP1. [ABSTRACT FROM AUTHOR]

Background: Plasmodium falciparum variant surface antigens (VSAs) contribute to malaria pathogenesis by mediating cytoadhesion of infected red blood cells to the microvasculature endothelium. In this study, we investigated the association between anti-VSA antibodies and clinical outcome in a controlled human malaria infection (CHMI) study. Method: We used flow cytometry and ELISA to measure levels of IgG antibodies to VSAs of five heterologous and one homologous P. falciparum parasite isolates, and to two PfEMP1 DBLβ domains in blood samples collected a day before the challenge and 14 days after infection. We also measured the ability of an individual's plasma to inhibit the interaction between PfEMP1 and ICAM1 using competition ELISA. We then assessed the association between the antibody levels, function, and CHMI defined clinical outcome during a 21-day follow-up period post infection using Cox proportional hazards regression. Results: Antibody levels to the individual isolate VSAs, or to two ICAM1-binding DBLβ domains of PfEMP1, were not associated with a significantly reduced risk of developing parasitemia or of meeting treatment criteria after the challenge after adjusting for exposure. However, anti-VSA antibody breadth (i.e., cumulative response to all the isolates) was a significant predictor of reduced risk of requiring treatment [HR 0.23 (0.10-0.50) p= 0.0002]. Conclusion: The breadth of IgG antibodies to VSAs, but not to individual isolate VSAs, is associated with protection in CHMI. [ABSTRACT FROM AUTHOR]

الملخص: أهداف البحث: يعتبر مجال الرابط المشابه دوفي للبروتين١ في غشاء كريات الدم الحمراء للمتصورة المنجلية مسئولا عن الآليات المسببة للمراضة للملاريا الدماغية. والأشخاص الذين يعيشون في المناطق الموبوءة بالملاريا لديهم أجسام مضادة محددة تجاه مجال البروتين ١ في غشاء كريات الدم الحمراء للمتصورة المنجلية والمناعة للتحكم في شدة الملاريا. يجعل هذا الميل مجال البروتين ١ في غشاء كريات الدم الحمراء للمتصورة المنجلية لقاح محتمل قائم على البروتين. وتهدف هذه الدراسة لاستكشاف الاستجابات المناعية الخلطية والخلوية الناجمة عن مجال الرابط المشابه دوفي ٢ بيتا- للبروتين ١ في غشاء كريات الدم الحمراء للمتصورة المنجلية للبروتين المؤتلف من عزل المتصورة المنجلية الإندونيسية. طرق البحث: تم التعبير عن البروتين المؤتلف في الإشريكية القولونية كأجزاء قابلة للذوبان وغير قابلة للذوبان، وهذا البروتين تمت تنقيته باستخدام التقارب اللوني قبل الحقن تحت الجلد لفئران ويستر في الأيام ١ و٢١و٤٢. تم حصاد المصل بعد ١٤ يوما من الحقنة الثانية والثالثة لقياس معيار تركيز الغلوبيولين المناعي IgG وCD4+ الخلايا بواسطة تقنية إليزا. النتائج: وجدنا أن تركيزات الغلوبيولين المناعي IgG وCD4+ الخلايا تزيد بعد الحقنة الثانية والحقن الثالثة. أظهر اختبار مان- ويتني اختلافا كبيرا بين مجموعات التحكم والعلاج لكلا من الغلوبيولين المناعي IgG وCD4+ الخلايا. وأظهر مزيدا من التحليل باستخدام الوصمة الغربية جسما مضادا محدد ضد مجال الرابط المشابه دوفي ٢ بيتا- للبروتين ١ في غشاء كريات الدم الحمراء للمتصورة المنجلية للبروتين المؤتلف. الاستنتاجات: يمكن لمجال الرابط المشابه دوفي ٢ بيتا- للبروتين ١ في غشاء كريات الدم الحمراء للمتصورة المنجلية للبروتين المؤتلف من عزل المتصورة المنجلية الإندونيسية أن يحفز استجابة مناعية خلطية وخلوية. المزيد من الدراسة على الغلوبيولين المناعي IgG التي تحتمل أن يكون لها تأثير مثبط ودور CD4+ الخلايا وعلاقتهم مع الخلايا المستجيبة الأخرى تعتبر أساسية لتحديد فعالية مجال الرابط المشابه دوفي ٢ بيتا- للبروتين ١ في غشاء كريات الدم الحمراء للمتصورة المنجلية كلقاح الملاريا المحتمل القائم على الببتيد. Abstract: Objectives: The Duffy binding-like (DBL) domain of the Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) is reportedly responsible for the pathophysiology of cerebral malaria. People living in endemic malaria areas possess specific antibodies against PfEMP1 and elicit immune responses to control the severity of malaria infection. Therefore, PfEMP1 may be a potential protein-based vaccine candidate. This study aimed to explore the humoral and cellular immune responses induced by the recombinant DBL2β-PfEMP1 obtained from the Indonesian P. falciparum isolate. Methods: The recombinant protein was expressed in Escherichia coli BL21(DE3) as soluble and insoluble fractions, and this protein was purified using affinity chromatography before administration as a subcutaneous injection in Wistar rats on days 1, 21, and 42. Sera were harvested 14 days after the second and third injections to determine the titre of IgG and the concentration of CD4+ cells using the enzyme linked immunosorbent assay (ELISA). Results: The IgG titre and the CD4+ cell concentration were found to be increased after the second and third injections. The Mann–Whitney test results showed a significant difference between the control and treatment groups for both the IgG and CD4+ cells (p = 0.001 and p = 0.000, respectively). Western blotting results indicated the presence of a specific antibody against the recombinant DBL2β-PfEMP1. Conclusions: The recombinant DBL2β-PfEMP1 of the Indonesian P. falciparum isolate could induce humoral and cellular immune responses. Further studies on IgG exerting inhibitory effects and the role of CD4+ cells and their association with other effector cells are essential to determine the efficacy of DBL2β-PfEMP1 and its potential application as a peptide-based malaria vaccine candidate.

Concerted efforts to fight malaria have caused significant reductions in global malaria cases and mortality. Sustaining this will be critical to avoid rebound and outbreaks of seasonal malaria. Identifying predictive attributes that define clinical malaria will be key to guide development of second-generation tools to fight malaria. Broadly reactive antibodies against variable surface antigens that are expressed on the surface of infected erythrocytes and merozoites stage antigens are targets of naturally acquired immunity and prime candidates for anti-malaria therapeutics and vaccines. However, predicting the relationship between the antigen-specific antibodies and protection from clinical malaria remains unresolved. Here, we used new datasets and multiple approaches combined with re-analysis of our previous data to assess the multi-dimensional and complex relationship between antibody responses and clinical malaria outcomes. We observed 22 antigens (17 PfEMP1 domains, 3 RIFIN family members, merozoite surface protein 3 (PF3D7_1035400), and merozoites-associated armadillo repeats protein (PF3D7_1035900) that were selected across three different clinical malaria definitions (1,000/2,500/5,000 parasites/µl plus fever). In addition, Principal Components Analysis (PCA) indicated that the first three components (Dim1, Dim2 and Dim3 with eigenvalues of 306, 48, and 29, respectively) accounted for 66.1% of the total variations seen. Specifically, the Dim1, Dim2 and Dim3 explained 52.8%, 8.2% and 5% of variability, respectively. We further observed a significant relationship between the first component scores and age with antibodies to PfEMP1 domains being the key contributing variables. This is consistent with a recent proposal suggesting that there is an ordered acquisition of antibodies targeting PfEMP1 proteins. Thus, although limited, and further work on the significance of the selected antigens will be required, these approaches may provide insights for identification of drivers of naturally acquired protective immunity as well as guide development of additional tools for malaria elimination and eradication.

Background: Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) is an important protein responsible for the pathogenesis of severe malaria, including cerebral malaria. The protein is highly diverse. The CIDR1α-PfEMP1 binds endothelial protein receptor (EPCR) and may associated with the brain swelling in childhood malaria. Objective: To analyze the CIDR1α-PfEMP1 from Indonesian isolate and determine its association with cerebral malaria outcome. Methods: Fifteen blood samples of clinically mild to severe malaria-patient were collected for DNA extraction. Malaria diagnosis was conducted microscopically by Giemsa-stained thin blood smear. The CIDR1α domain was amplified by PCR using specific primer and PCR product was sequenced. The nucleotide sequences were analyzed by NCBI blast, DNASIS MAX 3 and translated into amino acid sequences using Expasy Translation Tool. Results: One out of fifteen samples was severe malaria case and infected with P. falciparum, the rest were clinically mild to moderate malaria and infected with pure P. falciparum or mixed infection of P. falciparum and P. vivax. Amplification for CIDR1α domain resulted a single band of + 550 bp from a severe sample only. Sequencing of PCR product on both strands read 524 nucleotides and BLAST analysis confirmed as CIDR1α sequence. Multiple alignment showed 74-78% nucleotide sequence similarity with reference sequences, but amino acid sequences presented 23.5% homologous. Conclusion: An identified CIDR1α domain only from severe case implicating the potential association with the severe outcome including cerebral malaria, but the highly diverse of the domain needs further studies on the interaction with the pathological-causing receptor in the host.

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. [ABSTRACT FROM AUTHOR]

Background: There is a clear need for novel approaches to malaria vaccine development. We aimed to develop a genetically attenuated blood-stage vaccine and test its safety, infectivity, and immunogenicity in healthy volunteers. Our approach was to target the gene encoding the knob-associated histidine-rich protein (KAHRP), which is responsible for the assembly of knob structures at the infected erythrocyte surface. Knobs are required for correct display of the polymorphic adhesion ligand P. falciparum erythrocyte membrane protein 1 (PfEMP1), a key virulence determinant encoded by a repertoire of var genes.Methods: The gene encoding KAHRP was deleted from P. falciparum 3D7 and a master cell bank was produced in accordance with Good Manufacturing Practice. Eight malaria naïve males were intravenously inoculated (day 0) with 1800 (2 subjects), 1.8 × 105 (2 subjects), or 3 × 106 viable parasites (4 subjects). Parasitemia was measured using qPCR; immunogenicity was determined using standard assays. Parasites were rescued into culture for in vitro analyses (genome sequencing, cytoadhesion assays, scanning electron microscopy, var gene expression).Results: None of the subjects who were administered with 1800 or 1.8 × 105 parasites developed parasitemia; 3/4 subjects administered 3× 106 parasites developed significant parasitemia, first detected on days 13, 18, and 22. One of these three subjects developed symptoms of malaria simultaneously with influenza B (day 17; 14,022 parasites/mL); one subject developed mild symptoms on day 28 (19,956 parasites/mL); and one subject remained asymptomatic up to day 35 (5046 parasites/mL). Parasitemia rapidly cleared with artemether/lumefantrine. Parasitemia induced a parasite-specific antibody and cell-mediated immune response. Parasites cultured ex vivo exhibited genotypic and phenotypic properties similar to inoculated parasites, although the var gene expression profile changed during growth in vivo.Conclusions: This study represents the first clinical investigation of a genetically attenuated blood-stage human malaria vaccine. A P. falciparum 3D7 kahrp- strain was tested in vivo and found to be immunogenic but can lead to patent parasitemia at high doses.Trial Registration: Australian New Zealand Clinical Trials Registry (number: ACTRN12617000824369 ; date: 06 June 2017). [ABSTRACT FROM AUTHOR]

Abstract Background The pathogenesis of Plasmodium falciparum malaria is related to the ability of parasite‑infected 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.

Abstract Background During the erythrocytic cycle, Plasmodium falciparum malaria parasites express P. falciparum Erythrocyte Membrane Protein 1 (PfEMP1) that anchor the infected erythrocytes (IE) to the vascular lining of the host. The CIDRα1 domain of PfEMP1 is responsible for binding host endothelial protein C receptor (EPCR), and increasing evidence support that this interaction triggers severe malaria, accounting for the majority of malaria-related deaths. In high transmission regions, children develop immunity to severe malaria after the first few infections. This immunity is believed to be mediated by antibodies targeting and inhibiting PfEMP1, causing infected erythrocytes to circulate and be cleared in the spleen. The development of immunity to malaria coincides with acquisition of broad antibody reactivity across the CIDRα1 protein family. Altogether, this identifies CIDRα1 as an important vaccine target. However, the antigenic diversity of the CIDRα1 domain family is a challenge for vaccine development. Methods Immune responses in mice vaccinated with Virus-Like Particles (VLP) presenting CIDRα1 antigens were investigated. Antibody reactivity was tested to a panel of recombinant CIDRα1 domains, and the antibodies ability to inhibit EPCR binding by the recombinant CIDRα1 domains was tested in Luminex-based multiplex assays. Results VLP-presented CIDRα1.4 antigens induced a rapid and strong IgG response capable of inhibiting EPCR-binding of multiple CIDRα1 domains mainly within the group A CIDRα1.4–7 subgroups. Conclusions The study observations mirror those from previous CIDRα1 vaccine studies using other vaccine constructs and platforms. This suggests that broad CIDRα1 antibody reactivity may be achieved through vaccination with a limited number of CIDRα1 variants. In addition, this study suggest that this may be achieved through vaccination with a human compatible VLP vaccine platform.

ABSTRACT var genes encode Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP1) antigens. These highly diverse antigens are displayed on the surface of infected erythrocytes and play a critical role in immune evasion and sequestration of infected erythrocytes. Studies of var expression using non-leukocyte-depleted blood are challenging because of the predominance of host genetic material and lack of conserved var segments. Our goal was to enrich for parasite RNA, allowing de novo assembly of var genes and detection of expressed novel variants. We used two overall approaches: (i) enriching for total mRNA in the sequencing library preparations and (ii) enriching for parasite RNA with a custom capture array based on Roche’s SeqCap EZ enrichment system. The capture array was designed with probes based on the whole 3D7 reference genome and an additional >4,000 full-length var gene sequences from other P. falciparum strains. We tested each method on RNA samples from Malian children with severe or uncomplicated malaria infections. All reads mapping to the human genome were removed, the remaining reads were assembled de novo into transcripts, and from these, var-like transcripts were identified and annotated. The capture array produced the longest maximum length and largest numbers of var gene transcripts in each sample, particularly in samples with low parasitemia. Identifying the most-expressed var gene sequences in whole-blood clinical samples without the need for extensive processing or generating sample-specific reference genome data is critical for understanding the role of PfEMP1s in malaria pathogenesis. IMPORTANCE Malaria parasites display antigens on the surface of infected red blood cells in the human host that facilitate attachment to blood vessels, contributing to the severity of infection. These antigens are highly variable, allowing the parasite to evade the immune system. Identifying these expressed antigens is critical to understanding the development of severe malarial disease. However, clinical samples contain limited amounts of parasite genetic material, a challenge for sequencing efforts further compounded by the extreme diversity of the parasite surface antigens. We present a method that enriches for these antigen sequences in clinical samples using a custom capture array, requiring minimal processing in the field. While our results are focused on the malaria parasite Plasmodium falciparum, this approach has broad applicability to other highly diverse antigens from other parasites and pathogens such as those that cause giardiasis and leishmaniasis.

ABSTRACT Malaria parasites induce morphological and biochemical changes in the membranes of parasite-infected red blood cells (iRBCs) for propagation. Artemisinin combination therapies are the first-line antiplasmodials in countries of endemicity. However, the mechanism of action of artemisinin is unclear, and drug resistance decreases long-term efficacy. To understand whether artemisinin targets or interacts with iRBC membrane proteins, this study investigated the molecular changes caused by dihydroartemisinin (DHA), an artemisinin derivative, in Plasmodium falciparum 3D7 using a combined transcriptomic and membrane proteomic profiling approach. Optical microscopy and scanning electron microscopy showed that DHA can cause morphological variation in the iRBC membrane. We identified 125 differentially expressed membrane proteins, and functional analysis indicated structural molecule activity and protein export as key biological functions of the two omics studies. DHA treatment decreased the expression of var gene variants PF3D7_0415700 and PF3D7_0900100 dose-dependently. Western blotting and immunofluorescence analysis showed that DHA treatment downregulates the var gene encoding P. falciparum erythrocyte membrane protein-1 (pfEMP1). pfEMP1 knockout significantly increased artemisinin sensitivity. Results showed that pfEMP1 might be involved in the antimalarial mechanism of action of DHA and pfEMP1 or its regulated factors may be further exploited in antiparasitic drug design. The findings are beneficial for elucidating the potential effects of DHA on iRBC membrane proteins and developing new drugs targeting iRBC membrane. IMPORTANCE Malaria parasites induce morphological and biochemical changes in the membranes of parasite-infected red blood cells (iRBCs) for propagation, with artemisinin combination therapies as the first-line treatments. To understand whether artemisinin targets or interacts with iRBC membrane proteins, this study investigated the molecular changes caused by dihydroartemisinin (DHA), an artemisinin derivative, in Plasmodium falciparum 3D7 using a combined transcriptomic and membrane proteomic profiling approach. We found that DHA can cause morphological changes of iRBC membrane. Structural molecule activity and protein export are considered to be the key biological functions based on the two omics studies. pfEMP1 might be involved in the DHA mechanism of action. pfEMP1 or its regulated factors may be further exploited in antiparasitic drug design. The findings are beneficial for elucidating the potential effects of DHA on iRBC membrane proteins and developing new antimalarial drugs targeting iRBC membrane.

Strain-transcending antibodies against virulence-associated subsets of P. falciparum -infected erythrocyte surface antigens could protect children from severe malaria. However, the evidence supporting the existence of such antibodies is incomplete and inconsistent. One subset of surface antigens associated with severe malaria, rosette-mediating Plasmodium falciparum Erythrocyte Membrane Protein one (PfEMP1) variants, cause infected erythrocytes to bind to uninfected erythrocytes to form clusters of cells (rosettes) that contribute to microvascular obstruction and pathology. Here, we tested plasma from 80 individuals living in malaria-endemic regions for IgG recognition of the surface of four P. falciparum rosetting strains using flow cytometry. Broadly reactive plasma samples were then used in antibody elution experiments in which intact IgG was eluted from the surface of infected erythrocytes and transferred to heterologous rosetting strains to look for strain-transcending antibodies. We found that seroprevalence (percentage of positive plasma samples) against allopatric rosetting strains was high in adults (63%-93%) but lower in children (13%-48%). Strain-transcending antibodies were present in nine out of eleven eluted antibody experiments, with six of these recognizing multiple heterologous rosetting parasite strains. One eluate had rosette-disrupting activity against heterologous strains, suggesting PfEMP1 as the likely target of the strain-transcending antibodies. Naturally acquired strain-transcending antibodies to rosetting P. falciparum strains in humans have not been directly demonstrated previously. Their existence suggests that such antibodies could play a role in clinical protection and raises the possibility that conserved epitopes recognized by strain-transcending antibodies could be targeted therapeutically by monoclonal antibodies or vaccines., Competing Interests: The authors declare no conflict of interest.

The malaria vaccine is an important strategy for the global malaria elimination program, but the complexity of the Plasmodium antigen is a major hurdle in malaria vaccine development. The cysteine-rich interdomain region 1α (CIDR1α) of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is crucial in malaria pathogenesis, making it a vaccine candidate. This study investigated the leukocyte and IgM response generated after administering a CIDR1α-PfEMP1 recombinant protein injection in Wistar rats. The rats were divided into a control group, who received a physiological saline solution (PSS), and a treatment group, who were subcutaneously injected with 150 µg of purified CIDR1α-PfEMP1 protein three times at the 3-week interval. Blood samples were collected every week after each injection. The number of leukocytes were counted using a Neubauer chamber, and the IgM concentration was determined using an enzyme-linked immunosorbent assay (ELISA). Data were analyzed using an independent, paired-T test, a Mann–Whitney test, and a Wilcoxon test, based on the distribution of the data. The total number of leukocytes notably increased on day 29 (p < 0.05). The percentage of neutrophils decreased, especially on day 8 (p < 0.05), whereas the percentages of monocytes and lymphocytes increased, primarily on day 14 (p < 0.05). The IgM concentration increased on day 14 (p < 0.05). In conclusion, the CIDR1α-PfEMP1 recombinant protein may induce leukocyte and IgM responses, making it a potential malaria vaccine candidate.

Cytoadhesion of Plasmodium falciparum-infected erythrocytes (IEs) to the endothelial lining of blood vessels protects parasites from splenic destruction, but also leads to detrimental inflammation and vessel occlusion. Surface display of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) adhesion ligands exposes them to host antibodies and serum proteins. PfEMP1 are important targets of acquired immunity to malaria, and through evolution, the protein family has expanded and diversified to bind a select set of host receptors through antigenically diversified receptor-binding domains. Here, we show that complement component 1s (C1s) in serum cleaves PfEMP1 at semiconserved arginine motifs located at interdomain regions between the receptor-binding domains, rendering the IE incapable of binding the two main PfEMP1 receptors, CD36 and endothelial protein C receptor (EPCR). Bioinformatic analyses of PfEMP1 protein sequences from 15 P. falciparum genomes found the C1s motif was present in most PfEMP1 variants. Prediction of C1s cleavage and loss of binding to endothelial receptors was further corroborated by testing of several different parasite lines. These observations suggest that the parasites have maintained susceptibility for cleavage by the serine protease, C1s, and provides evidence for a complex relationship between the complement system and the P. falciparum cytoadhesion virulence determinant. [ABSTRACT FROM AUTHOR]

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Background: Loop-mediated isothermal amplification (LAMP) for malaria diagnosis at the point of care (POC) depends on the detection capacity of synthesized nucleic acids and the specificity of the amplification target. To improve malaria diagnosis, new colorimetric LAMP tests were developed using multicopy targets for Plasmodium vivax and Plasmodium falciparum detection. Methods: The cytochrome oxidase I (COX1) mitochondrial gene and the non-coding sequence Pvr47 for P. vivax, and the sub-telomeric sequence of erythrocyte membrane protein 1 (EMP1) and the non-coding sequence Pfr364 for P. falciparum were targeted to design new LAMP primers. The limit of detection (LOD) of each colorimetric LAMP was established and assessed with DNA extracted by mini spin column kit and the Boil & Spin method from 28 microscopy infections, 101 malaria submicroscopic infections detected by real-time PCR only, and 183 negatives infections by both microscopy and PCR. Results: The LODs for the colorimetric LAMPs were estimated between 2.4 to 3.7 parasites/µL of whole blood. For P. vivax detection, the colorimetric LAMP using the COX1 target showed a better performance than the Pvr47 target, whereas the Pfr364 target was the most specific for P. falciparum detection. All microscopic infections of P. vivax were detected by PvCOX1-LAMP using the mini spin column kit DNA extraction method and 81% (17/21) were detected using Boil & Spin sample preparation. Moreover, all microscopic infections of P. falciparum were detected by Pfr364-LAMP using both sample preparation methods. In total, PvCOX1-LAMP and Pfr364-LAMP detected 80.2% (81 samples) of the submicroscopic infections using the DNA extraction method by mini spin column kit, while 36.6% (37 samples) were detected using the Boil & Spin sample preparation method. Conclusion: The colorimetric LAMPs with multicopy targets using the COX1 target for P. vivax and the Pfr364 for P. falciparum have a high potential to improve POC malaria diagnosis detecting a greater number of submicroscopic Plasmodium infections. [ABSTRACT FROM AUTHOR]

Plasmodium falciparum pathogenesis is complex and intimately connected to vascular physiology. This is exemplified by cerebral malaria (CM), a neurovascular complication that accounts for most of the malaria deaths worldwide. P. falciparum sequestration in the brain microvasculature is a hallmark of CM and is not replicated in animal models. Numerous aspects of the disease are challenging to fully understand from clinical studies, such as parasite binding tropism or causal pathways in blood–brain barrier breakdown. Recent bioengineering approaches allow for the generation of 3D microvessels and organ-specific vasculature that provide precise control of vessel architecture and flow dynamics, and hold great promise for malaria research. Here, we discuss recent and future applications of bioengineered microvessels in malaria pathogenesis research. Research into cytoadhesive complications of Plasmodium falciparum infection has been hampered by the lack of suitable animal models and limitations of endothelial cell monolayer models. Bioengineered microvessels offer precise control over vascular cell types, branching architecture, lumen diameter, and flow dynamics. They provide new opportunities for investigating Plasmodium –vessel interactions. 3D microvessel models have been used to explore how flow dynamics, vessel diameter, and endothelial activation influence sequestration. They can also model the endothelial cells' response to host and parasite inflammatory products. Future bioengineered models could include tissue-specific vasculature to investigate organ-specific injury in severe malaria, evaluate novel therapeutic interactions, or improve our understanding of other key parasite–blood vessel interactions in the malaria life cycle. [ABSTRACT FROM AUTHOR]

Abstract Background Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP1) antigens play a critical role in host immune evasion. Serologic responses to these antigens have been associated with protection from clinical malaria, suggesting that antibodies to PfEMP1 antigens may contribute to natural immunity. The first N-terminal constitutive domain in a PfEMP1 is the Duffy binding-like alpha (DBL-α) domain, which contains a 300 to 400 base pair region unique to each particular protein (the DBL-α “tag”). This DBL-α tag has been used as a marker of PfEMP1 diversity and serologic responses in malaria-exposed populations. In this study, using sera from a malaria-endemic region, responses to DBL-α tags were compared to responses to the corresponding entire DBL-α domain (or “parent” domain) coupled with the succeeding cysteine-rich interdomain region (CIDR). Methods A protein microarray populated with DBL-α tags, the parent DBL-CIDR head structures, and downstream PfEMP1 protein fragments was probed with sera from Malian children (aged 1 to 6 years) and adults from the control arms of apical membrane antigen 1 (AMA1) vaccine clinical trials before and during a malaria transmission season. Serological responses to the DBL-α tag and the DBL-CIDR head structure were measured and compared in children and adults, and throughout the season. Results Malian serologic responses to a PfEMP1’s DBL-α tag region did not correlate with seasonal malaria exposure, or with responses to the parent DBL-CIDR head structure in either children or adults. Parent DBL-CIDR head structures were better indicators of malaria exposure. Conclusions Larger PfEMP1 domains may be better indicators of malaria exposure than short, variable PfEMP1 fragments such as DBL-α tags. PfEMP1 head structures that include conserved sequences appear particularly well suited for study as serologic predictors of malaria exposure.

Plasmodium falciparum-infected erythrocytes (PfIEs) adhere to endothelial cell receptors (ECRs) of blood vessels mainly via PfEMP1 proteins to escape elimination via the spleen. Evidence suggests that P. vivax-infected reticulocytes (PvIRs) also bind to ECRs, presumably enabled by VIR proteins, as shown by inhibition experiments and studies with transgenic P. falciparum expressing vir genes. To test this hypothesis, our study investigated the involvement of VIR proteins in cytoadhesion using vir gene-expressing P. falciparum transfectants. Those VIR proteins with a putative transmembrane domain were present in Maurer’s clefts, and some were also present in the erythrocyte membrane. The VIR protein without a transmembrane domain (PVX_050690) was not exported. Five of the transgenic P. falciparum cell lines, including the one expressing PVX_050690, showed binding to CD36. We observed highly increased expression of specific var genes encoding PfEMP1s in all CD36-binding transfectants. These results suggest that ectopic vir expression regulates var expression through a yet unknown mechanism. In conclusion, the observed cytoadhesion of P. falciparum expressing vir genes depended on PfEMP1s, making this experimental unsuitable for characterizing VIR proteins.

Sequestration of Plasmodium falciparum(P. falciparum)-infected erythrocytes to host endothelium through the parasite-derived P. falciparum erythrocyte membrane protein 1 (PfEMP1) adhesion proteins is central to the development of malaria pathogenesis. PfEMP1 proteins have diversified and expanded to encompass many sequence variants, conferring each parasite a similar array of human endothelial receptor-binding phenotypes. Here, we analyzed RNA-seq profiles of parasites isolated from 32 P. falciparum-infected adult travellers returning to Germany. Patients were categorized into either malaria naive (n = 15) or pre-exposed (n = 17), and into severe (n = 8) or non-severe (n = 24) cases. For differential expression analysis, PfEMP1-encoding var gene transcripts were de novo assembled from RNA-seq data and, in parallel, var-expressed sequence tags were analyzed and used to predict the encoded domain composition of the transcripts. Both approaches showed in concordance that severe malaria was associated with PfEMP1 containing the endothelial protein C receptor (EPCR)-binding CIDRα1 domain, whereas CD36-binding PfEMP1 was linked to non-severe malaria outcomes. First-time infected adults were more likely to develop severe symptoms and tended to be infected for a longer period. Thus, parasites with more pathogenic PfEMP1 variants are more common in patients with a naive immune status, and/or adverse inflammatory host responses to first infections favor the growth of EPCR-binding parasites.

Over 30 million women living in P. falciparum endemic areas are at risk of developing malaria during pregnancy every year. Placental malaria is characterized by massive accumulation of infected erythrocytes in the intervillous space of the placenta, accompanied by infiltration of immune cells, particularly monocytes. The consequent local inflammation and the obstruction of the maternofetal exchanges can lead to severe clinical outcomes for both mother and child. Even if protection against the disease can gradually be acquired following successive pregnancies, the malaria parasite has developed a large panel of evasion mechanisms to escape from host defense mechanisms and manipulate the immune system to its advantage. Infected erythrocytes isolated from placentas of women suffering from placental malaria present a unique phenotype and express the pregnancy-specific variant VAR2CSA of the Plasmodium falciparum Erythrocyte Membrane Protein (PfEMP1) family at their surface. The polymorphic VAR2CSA protein is able to mediate the interaction of infected erythrocytes with a variety of host cells including placental syncytiotrophoblasts and leukocytes but also with components of the immune system such as non-specific IgM. This review summarizes the described VAR2CSA-mediated host defense evasion mechanisms employed by the parasite during placental malaria to ensure its survival and persistence.

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.

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. [ABSTRACT FROM AUTHOR]

Over 30 million women living in P. falciparum endemic areas are at risk of developing malaria during pregnancy every year. Placental malaria is characterized by massive accumulation of infected erythrocytes in the intervillous space of the placenta, accompanied by infiltration of immune cells, particularly monocytes. The consequent local inflammation and the obstruction of the maternofetal exchanges can lead to severe clinical outcomes for both mother and child. Even if protection against the disease can gradually be acquired following successive pregnancies, the malaria parasite has developed a large panel of evasion mechanisms to escape from host defense mechanisms and manipulate the immune system to its advantage. Infected erythrocytes isolated from placentas of women suffering from placental malaria present a unique phenotype and express the pregnancy-specific variant VAR2CSA of the Plasmodium falciparum Erythrocyte Membrane Protein (PfEMP1) family at their surface. The polymorphic VAR2CSA protein is able to mediate the interaction of infected erythrocytes with a variety of host cells including placental syncytiotrophoblasts and leukocytes but also with components of the immune system such as non-specific IgM. This review summarizes the described VAR2CSA-mediated host defense evasion mechanisms employed by the parasite during placental malaria to ensure its survival and persistence. [ABSTRACT FROM AUTHOR]

Plasmodium falciparum extensively remodels host cells by translocating numerous proteins into the cytoplasm of red blood cells (RBCs) after invasion. Among these exported proteins, members of the Plasmodium helical interspersed subtelomeric (PHIST) family are crucial for host cell remodeling and host-parasite interactions, and thereby contribute to malaria pathogenesis. Herein, we explored the function of PF3D7_1372300, a member of the PHIST/PHISTa-like subfamily. PF3D7_1372300 was highly transcribed and expressed during the blood stage of P. falciparum, and distributed throughout RBCs, but most abundant at the erythrocyte membrane. Specific interaction of PF3D7_1372300 with the cytoplasmic tail of P. falciparum erythrocyte membrane protein 1 (PfEMP1) was revealed by immunofluorescence assay, in vitro intermolecular interaction assays. The interaction sites of PF3D7_1372300 with PfEMP1 ATS domain were found involved more than 30 amino acids (aa) at several positions. The findings deepen our understanding of host-parasite interactions and malaria pathogenesis.

Plasmodium falciparum extensively remodels host cells by translocating numerous proteins into the cytoplasm of red blood cells (RBCs) after invasion. Among these exported proteins, members of the Plasmodium helical interspersed subtelomeric (PHIST) family are crucial for host cell remodeling and host-parasite interactions, and thereby contribute to malaria pathogenesis. Herein, we explored the function of PF3D7_1372300, a member of the PHIST/PHISTa-like subfamily. PF3D7_1372300 was highly transcribed and expressed during the blood stage of P. falciparum , and distributed throughout RBCs, but most abundant at the erythrocyte membrane. Specific interaction of PF3D7_1372300 with the cytoplasmic tail of P. falciparum erythrocyte membrane protein 1 (PfEMP1) was revealed by immunofluorescence assay, in vitro intermolecular interaction assays. The interaction sites of PF3D7_1372300 with PfEMP1 ATS domain were found involved more than 30 amino acids (aa) at several positions. The findings deepen our understanding of host-parasite interactions and malaria pathogenesis. [ABSTRACT FROM AUTHOR]

Malaria remains a significant contributor to the global burden of disease, with around 40% of the world’s population at risk of Plasmodium infections. The development of an effective vaccine against the malaria parasite would mark a breakthrough in the fight to eradicate the disease. Over time, natural infection elicits a robust immune response against the blood stage of the parasite, providing protection against malaria. In recent years, we have gained valuable insight into the mechanisms by which IgG acts to prevent pathology and inhibit parasite replication, as well as the potential role of immunoglobulin M (IgM) in these processes. Here, we discuss recent advances in our understanding of the mechanisms, acquisition, and maintenance of naturally acquired immunity, and the relevance of these discoveries for the development of a potential vaccine against the blood stage of Plasmodium falciparum.

ABSTRACT Mary Lopez-Perez works on immunology and pathogenesis of malaria. In this mSphere of Influence article, she reflects on how the paper “Functional antibodies against VAR2CSA in nonpregnant populations from Colombia exposed to Plasmodium falciparum and Plasmodium vivax” by S. Gnidehou, J. Doritchamou, E. M. Arango, A. Cabrera, et al. (Infect Immun 82:2565−2573, 2014, https://doi.org/10.1128/IAI.01594-14) made her cautious of relying exclusively on recombinant proteins when evaluating antibody responses.

There is introduced the analysis of molecular-genetic mechanisms of tropical malaria pathogenesis and P. falciparum virulence. It is shown, that pathogenesis of tropical malaria is associated with the properties of red blood cells membrane surface (RBCs or erythrocytes) that are infected by P. falciparum. There are «knobs structures» on membrane surface infected RBCs. Knobs structures contains a complex of P. falciparum proteins – PfEMP1 (Plasmodium falciparum erythrocyte membrane protein 1). PfEMP1 is associated with virulence of P. falciparum. Complex PfEMP1 has difficult polymorphous structure. Domains of PfEMP1 are able to associate with different cell receptors. Virulence`s individual components of the main factor are selectively sensitive to different tissues and organs. The severity of the clinical malaria infection course depends on the complex structure PfEMP1 of malaria parasites. Composition of polypeptide PfEMP1 is determined by var-complex. Nowadays there are 60 variants of var-complex. Regulation of gene expression, forming part of the var-complex, is carried out on a molecular-genetic level, cellular level, tissue level. Modern research in this area are aimed to explore genes polymorphism of the virulence`s main factor, to identify mechanism of its differential expression. Search of molecular – genetic markers is relevant to develop methods of gene diagnostic and malaria vaccine.

Abstract Background Antibody immunity is thought to be essential to prevent severe Plasmodium falciparum infection, but the exact correlates of protection are unknown. Over time, children in endemic areas acquire non-sterile immunity to malaria that correlates with development of antibodies to merozoite invasion proteins and parasite proteins expressed on the surface of infected erythrocytes. Results A 1000 feature P. falciparum 3D7 protein microarray was used to compare P. falciparum-specific seroreactivity during acute infection and 30 days after infection in 23 children with uncomplicated malaria (UM) and 25 children with retinopathy-positive cerebral malaria (CM). All children had broad P. falciparum antibody reactivity during acute disease. IgM reactivity decreased and IgG reactivity increased in convalescence. Antibody reactivity to CIDR domains of “virulent” PfEMP1 proteins was low with robust reactivity to the highly conserved, intracellular ATS domain of PfEMP1 in both groups. Although children with UM and CM differed markedly in parasite burden and PfEMP1 exposure during acute disease, neither acute nor convalescent PfEMP1 seroreactivity differed between groups. Greater seroprevalence to a conserved Group A-associated ICAM binding extracellular domain was observed relative to linked extracellular CIDRα1 domains in both case groups. Pooled immune IgG from Malawian adults revealed greater reactivity to PfEMP1 than observed in children. Conclusions Children with uncomplicated and cerebral malaria have similar breadth and magnitude of P. falciparum antibody reactivity. The utility of protein microarrays to measure serological recognition of polymorphic PfEMP1 antigens needs to be studied further, but the study findings support the hypothesis that conserved domains of PfEMP1 are more prominent targets of cross reactive antibodies than variable domains in children with symptomatic malaria. Protein microarrays represent an additional tool to identify cross-reactive Plasmodium antigens including PfEMP1 domains that can be investigated as strain-transcendent vaccine candidates.