Erythrocyte Binding Antigen Research Articles

Contribution of Functional Antimalarial Immunity to Measures of Parasite Clearance in Therapeutic Efficacy Studies of Artemisinin Derivatives.

BackgroundAntibodies to the blood stages of malaria parasites enhance parasite clearance and antimalarial efficacy. The antibody subclass and functions that contribute to parasite clearance during antimalarial treatment and their relationship to malaria transmission intensity have not been characterized.MethodsLevels of immunoglobulin G (IgG) subclasses and C1q fixation in response to Plasmodium falciparum merozoite antigens (erythrocyte-binding antigen [EBA] 175RIII-V, merozoite surface protein 2 [MSP-2], and MSP-142) and opsonic phagocytosis of merozoites were measured in a multinational trial assessing the efficacy of artesunate therapy across 11 Southeast Asian sites. Regression analyses assessed the effects of antibody seropositivity on the parasite clearance half-life (PC½), having a PC½ of ≥5 hours, and having parasitemia 3 days after treatment.ResultsIgG3, followed by IgG1, was the predominant IgG subclass detected (seroprevalence range, 5%–35% for IgG1 and 27%–41% for IgG3), varied across study sites, and was lowest in study sites with the lowest transmission intensity and slowest mean PC½. IgG3, C1q fixation, and opsonic-phagocytosis seropositivity were associated with a faster PC½ (range of the mean reduction in PC½, 0.47–1.16 hours; P range, .001–.03) and a reduced odds of having a PC½ of ≥5 hours and having parasitemia 3 days after treatment.ConclusionsThe prevalence of IgG3, complement-fixing antibodies, and merozoite phagocytosis vary according to transmission intensity, are associated with faster parasite clearance, and may be sensitive surrogates of an augmented clearance capacity of infected erythrocytes. Determining the functional immune mechanisms associated with parasite clearance will improve characterization of artemisinin resistance.

The impact of early life exposure to Plasmodium falciparum on the development of naturally acquired immunity to malaria in young Malawian children

BackgroundAntibodies targeting malaria blood-stage antigens are important targets of naturally acquired immunity, and may act as valuable biomarkers of malaria exposure.MethodsSix-hundred and one young Malawian children from a randomized trial of prenatal nutrient supplementation with iron and folic acid or pre- and postnatal multiple micronutrients or lipid-based nutrient supplements were followed up weekly at home and febrile episodes were investigated for malaria from birth to 18 months of age. Antibodies were measured for 601 children against merozoite surface proteins (MSP1 19kD, MSP2), erythrocyte binding antigen 175 (EBA175), reticulocyte binding protein homologue 2 (Rh2A9), schizont extract and variant surface antigens expressed by Plasmodium falciparum-infected erythrocytes (IE) at 18 months of age. The antibody measurement data was related to concurrent malaria infection and to documented episodes of clinical malaria.ResultsAt 18 months of age, antibodies were significantly higher among parasitaemic than aparasitaemic children. Antibody levels against MSP1 19kD, MSP2, schizont extract, and IE variant surface antigens were significantly higher in children who had documented episodes of malaria than in children who did not. Antibody levels did not differ between children with single or multiple malaria episodes before 18 months, nor between children who had malaria before 6 months of age or between 6 and 18 months.ConclusionsAntibodies to merozoite and IE surface antigens increased following infection in early childhood, but neither age at first infection nor number of malaria episodes substantially affected antibody acquisition. These findings have implications for malaria surveillance during early childhood in the context of elimination.Trials registration Clinical Trials Registration: NCT01239693 (Date of registration: 11-10-2010). URL: http://www.ilins.org

Shed EBA-175 mediates red blood cell clustering that enhances malaria parasite growth and enables immune evasion.

Erythrocyte Binding Antigen of 175 kDa (EBA-175) has a well-defined role in binding to glycophorin A (GpA) during Plasmodium falciparum invasion of erythrocytes. However, EBA-175 is shed post invasion and a role for this shed protein has not been defined. We show that EBA-175 shed from parasites promotes clustering of RBCs, and EBA-175-dependent clusters occur in parasite culture. Region II of EBA-175 is sufficient for clustering RBCs in a GpA-dependent manner. These clusters are capable of forming under physiological flow conditions and across a range of concentrations. EBA-175-dependent RBC clustering provides daughter merozoites ready access to uninfected RBCs enhancing parasite growth. Clustering provides a general method to protect the invasion machinery from immune recognition and disruption as exemplified by protection from neutralizing antibodies that target AMA-1 and RH5. These findings provide a mechanistic framework for the role of shed proteins in RBC clustering, immune evasion, and malaria.

Characterization of anti-EBA175RIII-V in asymptomatic adults and children living in communities in the Greater Accra Region of Ghana with varying malaria transmission intensities

BackgroundAntibodies against Region III-V of the erythrocyte binding antigen (EBA) 175 (EBA175RIII-V) have been suggested to provide protection from malaria in a natural infection. However, the quality and quantity of naturally induced antibodies to EBA175RIII-V has not been fully characterized in different cohorts of Ghanaians. This study sought to determine the characteristics of antibodies against EBA175RIII-V in asymptomatic adults and children living in two communities of varying P. falciparum parasite prevalence in southern Ghana.MethodsMicroscopic evaluation of thick and thin blood smears was used to identify asymptomatic Plasmodium falciparum carriage and indirect enzyme linked immunosorbent (ELISA) used to assess antibody concentrations and avidity.ResultsParasite carriage estimated by microscopy in Obom was 35.6% as opposed to 3.5% in Asutsuare. Levels of IgG, IgG1, IgG2, IgG3 and IgG4 against EBA175RIII-V in the participants from Obom were significantly higher (P < 0.05, Dunn’s Multiple Comparison test) than those in Asutsuare. However the relative avidity of IgG antibodies against EBA175RIII-V was significantly higher (P < 0.0001, Mann Whitney test) in Asutsuare than in Obom.ConclusionsPeople living in communities with limited exposure to P. falciparum parasites have low quantities of high avidity antibodies against EBA175RIII-V whilst people living in communities with high exposure to the parasites have high quantities of age-dependent but low avidity antibodies against EBA175RIII-V.

Assessing naturally acquired immune response and malaria treatment outcomes in Lagos, Nigeria

Background: There are emerging reports of poor efficacy of artemisinin-based combination treatment (ACT). However, mutations on the Kelch-13 gene marking delayed parasite clearance have no clinically defined relationship with ACT resistance across Africa. With increasing malaria control efforts, declining acquired immunity could be responsible for varying drug response profiles that may be dependent on levels of exposure to infections. To examine antibody responses against malaria and the influence on the efficacy of artemether-lumefantrine (AL), plasma samples were collected, prior to treatment, from individuals presenting with uncomplicated malaria. Methods: Participants were stratified into two groups: early (within 24 hours, N = 20) and late (between 48 – 72 hours, N = 30) parasite clearance after treatment, as determined by var gene acidic terminal sequence (varATS) polymerase chain reaction. Magnetic bead-based luminex assay was used to profile antibody responses specific to a panel of 21 Plasmodium falciparum sporozoite, merozoite and An. gambiae salivary antigens. Results: Median fluorescence intensity (MFI) of the antibodies was highest against glutamate-rich protein (GLURP-R0) and lowest against merozoite surface protein (MSP2) antigen. Analysis showed a positive correlation between expression of immunity and age of individuals (P = 0.023). However, there was no association between parasite density and antibody responses, except a significant positive relationship with reticulocyte binding protein-like homologue 5 (Rh5), P = 0.047; Plasmodium exported protein (Hyp2), P = 0.037 and merozoite surface protein 11 (H103), P = 0.038. Though higher levels of antibodies against erythrocyte binding antigens (EBA 140 and 175), MSP1.19, GLURP, circumsporozoite protein (CSP) and Rh4.2 were observed in individuals who recorded early parasite clearance, there was no significant difference in antibody responses in the early and late parasitological response groups. Conclusions: Characterization of additional markers in larger populations is required to reveal potential immunological correlates of drug efficacy.

Effect of nutrient supplementation on the acquisition of humoral immunity to Plasmodium falciparum in young Malawian children

BackgroundThere is evidence that suggests that undernutrition has a detrimental effect on malarial immunity in children. The aim of the study was to discover whether nutrient supplementation improved development of malarial antibody immunity in children up to 18 months of age.MethodsThe study was conducted with a subset of 432 Malawian children from a randomized controlled trial of nutritional supplements. The arms included pre- and postnatal small-quantity lipid-based nutrient supplements for both mother and child; prenatal supplementation with iron and folic acid; and pre- and postnatal supplementation with multiple micronutrients. Paired plasma samples were collected at 6 and 18 months of age. The levels of antibodies against merozoite surface protein 1 (MSP1 19kD) and MSP2, erythrocyte binding antigen 175 (EBA175), reticulocyte binding protein homologue 2A (Rh2A9), schizont extract and variant antigens expressed on the surface of infected erythrocytes were measured.ResultsAt 18 months of age, 5.4% of children were parasitaemic by microscopy and 49.1% were anaemic. Antibodies to the tested merozoite antigens and schizont extract increased between 6 and 18 months and this increase was statistically significant for MSP1, MSP2 and EBA175 (p < 0.0001) whereas IgG to variant surface antigens decreased with increasing age (p < 0.0001). However, the supplementation type did not have any impact on the prevalence or levels of antibodies at either 6 or 18 months of age to any of the tested malaria antigens in either univariate analysis or multivariate analysis after adjusting for covariates.ConclusionsPre- and postnatal lipid-based nutrient supplementation did not alter malaria antibody acquisition during infancy, compared to prenatal supplementation with iron and folic acid or pre- and postnatal supplementation with multiple micronutrients.Trail registeration Clinicaltrials.gov registration number NCT01239693

Complex signatures of natural selection at GYPA.

The human MN blood group antigens are isoforms of glycophorin A (GPA) encoded by the gene, GYPA, and are the most abundant erythrocyte sialoglycoproteins. The distribution of MN antigens has been widely studied in human populations yet the evolutionary and/or demographic factors affecting population variation remain elusive. While the primary function of GPA is yet to be discovered, it serves as the major binding site for the 175-kD erythrocyte-binding antigen (EB-175) of the malarial parasite, Plasmodium falciparum, a major selective pressure in recent human history. More specifically, exon two of GYPA encodes the receptor-binding ligand to which P. falciparum binds. Accordingly, there has been keen interest in understanding what impact, if any, natural selection has had on the distribution of variation in GYPA and exon two in particular. To this end, we resequenced GYPA in individuals sampled from both P. falciparum endemic (sub-Saharan Africa and South India) and non-endemic (Europe and East Asia) regions of the world. Observed patterns of variation suggest that GYPA has been subject to balancing selection in populations living in malaria endemic areas and in Europeans, but no such evidence was found in samples from East Asia, Oceania, and the Americas. These results are consistent with malaria acting as a selective pressure on GYPA, but also suggest that another selective force has resulted in a similar pattern of variation in Europeans. Accordingly, GYPA has perhaps a more complex evolutionary history, wherein on a global scale, spatially varying selective pressures have governed its natural history.

Possible Mimics of Duffy Binding Protein-II for &amp;lt;i&amp;gt;Plasmodium vivax&amp;lt;/i&amp;gt; Binding Endothelial Cells or Binding &amp;lt;i&amp;gt;Plasmodium falciparum&amp;lt;/i&amp;gt; by Mimicking Epitope on Erythrocyte Binding Antigen-175 A

Two molecules from the same source, Κ casein, are suggested as treatments to prevent infection by Plasmodium vivax, malaria virus, through the prevention of Duffy Binding Protein II (DBPII) monomers 1 and 2 from binding. By preventing DBPII monomer 1 and 2 binding there would be half the Duffy Antigen Receptor for Chemokines (DARC) binding to the DBPII trimer. This may prevent infection by this virus. Here the essential, structural, characterization of two potential binding mimics of the DBPII monomer 2 to prevent its binding to DBPII monomer 1, are provided. Κ casein is treated with NaBH4 in 1 N NH4OH (pH 11.4) to produce the two molecules; N-acetamido deoxy neuraminyl (α 2->3’) N-acetamido deoxy neuraminyl (α 2->6’) galactosyl 4’ (di-hydrido) sulfo (β 1->3) N-acetamido deoxy galactosaminyl 6 (di-hydrido) sulfo di-(hydrido) di-phospho serinyl (di-hydrido) sulfo tyrosine dipeptide and N-acetamido deoxy neuraminyl (α 2->3’) N-acetamido deoxy neuraminyl (α 2->6’) 4’ (di-hydrido) sulfo galactosyl (β 1-> 3) 6 (d-hydrido) sulfo 1,5 anhydro N-acetamido deoxy galactosaminitol. Only the first of the two molecules presents a (di-hydrido) sulfo tyrosine, yet both have two (di-hydrido) sulfo groups. Still the dipeptide may mimic another sulfo tyrosine on the DBPII monomer 2, thought to be away from T266 (threonine 266) a significant distance, possibly Y363 of DBPII monomer 2. These molecules may also mimic the appropriate epitope on Erythrocyte Binding Antigen (EBA)-175 of Plasmodium falciparum to prevent infection by this virus.

High Level of Specific Anti-Plasmodium Falciparum Merozoite IgG1 Antibodies in Rural Asymptomatic Individuals of Dienga, South-Eastern Gabon.

Plasmodium falciparum merozoite antigens (PfMAgs) play an essential role in the development of immunity to malaria. Currently, P. falciparum: protein 113 (Pf 113), apical membrane antigen 1 (AMA1), erythrocyte binding antigens (EBA175), and reticulocyte binding protein homologue 5 (RH5) are among the most PfMAgs studied. A comparative analysis of naturally acquired antibodies against these antigens in children would increase our knowledge about the development of protective immunity.Analysis of antibodies to Pf113, PfAMA1, PfEBA175, and PfRH5 was conducted in rural population during 2013 and 2014. Both prevalence and levels of total IgG anti-PfAMA1 were higher than that of IgG anti-PfEBA175, anti-PfRH5, and anti-Pf113. Seroconversion to PfAMA1 and PfEBA175 occurred moderately in young children and reached to the maximum in adolescent and in adults. High prevalence of IgG anti-Pf113 was observed in young children of 3 to 6 years old in 2013. The four antigens were recognized by IgG 1, 2, 3, and 4 antibodies from a large proportion of the subjects, and all of them induced high levels of specific IgG1 against PfAMA1, PfEBA175, fewer by Pf113 and PfRH5.Many asymptomatic children had specific IgG1 recognizing multiple antigens, and these IgG1 antibodies could be associated with a reduced risk of developing malaria symptoms.

Genetic structure of two erythrocyte binding antigens of Plasmodium falciparum reveals a contrasting pattern of selection

Erythrocyte binding antigens 175 (EBA-175) and 140 (EBA-140) play key roles in erythrocyte invasion by binding to glycophorin A (GPA) and C (GPC) respectively in human malaria. Since antigenic variation in malaria endemic region is a major barrier to development of effective vaccine, we explore the nature and pattern of sequence diversity of these two vaccine candidates in Kolkata, India. Population genetic parameters based on parasite sequences representing region II of Pfeba-175 and Pfeba-140 genes were estimated using DnaSP V.5.10 and MEGA version 6.0. A novel molecular docking approach was implemented to assess the binding affinities of Kolkata Pfeba-175 variants with GPA. P. falciparum Kolkata isolates experienced a recent population expansion as documented by negative Tajima's D, Fu & Li's statistics, unimodal mismatch distribution and star-like median-joining network for both loci. Positive selection seemed to play a major role in shaping the diversity of Pfeba-175 (dN/dS=2.45, and McDonald-Kreitman P-value=0.04) with successive accumulation of Q584K/E, E592A and R664S deriving high frequency haplotypes designated here as F2KH3 and F2KH1. In silico molecular docking demonstrated that polypeptides encoded by F2KH1 and F2KH3 were capable of engaging the parasite ligand into energetically favorable interaction with GPA. Our data demonstrated emergence of Pfeba-175 sequences harboring selectively advantageous nonsynonymous substitutions on Pf3D7 sequence background in the Kolkata parasite isolates. A contrasting pattern of Pf3D7-centric expansion of parasite sequences was noted for Pfeba-140. Together, this study provides a firm genetic and biological support favoring a dominant role of EBA-175 in erythrocyte invasion.

Synthetic Antigens Derived from Plasmodium falciparum Sporozoite, Liver, and Blood Stages: Naturally Acquired Immune Response and Human Leukocyte Antigen Associations in Individuals Living in a Brazilian Endemic Area.

Peptide vaccine strategies using Plasmodium-derived antigens have emerged as an attractive approach against malaria. However, relatively few studies have been conducted with malaria-exposed populations from non-African countries. Herein, the seroepidemiological profile against Plasmodium falciparum of naturally exposed individuals from a Brazilian malaria-endemic area against synthetic peptides derived from vaccine candidates circumsporozoite protein (CSP), liver stage antigen-1 (LSA-1), erythrocyte binding antigen-175 (EBA-175), and merozoite surface protein-3 (MSP-3) was investigated. Moreover, human leukocyte antigen (HLA)-DRB1* and HLA-DQB1* were evaluated to characterize genetic modulation of humoral responsiveness to these antigens. The study was performed using blood samples from 187 individuals living in rural malaria-endemic villages situated near Porto Velho, Rondônia State. Specific IgG and IgM antibodies and IgG subclasses were detected by enzyme-linked immunosorbent assay, and HLA-DRB1* and HLA-DQB1* low-resolution typing was performed by PCR-SSP. All four synthetic peptides were broadly recognized by naturally acquired antibodies. Regarding the IgG subclass profile, only CSP induced IgG1 and IgG3 antibodies, which is an important fact given that the acquisition of protective immunity appears to be associated with the cytophilicity of IgG1 and IgG3 antibodies. HLA-DRB1*11 and HLA-DQB1*7 had the lowest odds of responding to EBA-175. Our results showed that CSP, LSA-1, EBA, and MSP-3 are immunogenic in natural conditions of exposure and that anti-EBA antibody responses appear to be modulated by HLA class II antigens.

Acquisition, maintenance and adaptation of invasion inhibitory antibodies against Plasmodium falciparum invasion ligands involved in immune evasion.

Erythrocyte-binding antigens (EBAs) and P. falciparum reticulocyte-binding homologue proteins (PfRhs) are two important protein families that can vary in expression and utilization by P. falciparum to evade inhibitory antibodies. We evaluated antibodies at repeated time-points among individuals living in an endemic region in Nigeria over almost one year against these vaccine candidates. Antibody levels against EBA140, EBA175, EBA181, PfRh2, PfRh4, and MSP2, were measured by ELISA. We also used parasites with disrupted EBA140, EBA175 and EBA181 genes to show that all these were targets of invasion inhibitory antibodies. However, antigenic targets of inhibitory antibodies were not stable and changed substantially over time in most individuals, independent of age. Antibodies levels measured by ELISA also varied within and between individuals over time and the antibodies against EBA181, PfRh2 and MSP2 declined more rapidly in younger individuals (≤15 years) compared with older (>15). The breadth of high antibody responses over time was more influenced by age than by the frequency of infection. High antibody levels were associated with a more stable invasion inhibitory response, which could indicate that during the long process of formation of immunity, many changes not only in levels but also in functional responses are needed. This is an important finding in understanding natural immunity against malaria, which is essential for making an efficacious vaccine.

Pooled-DNA sequencing identifies genomic regions of selection in Nigerian isolates of Plasmodium falciparum

BackgroundThe burden of falciparum malaria is especially high in sub-Saharan Africa. Differences in pressure from host immunity and antimalarial drugs lead to adaptive changes responsible for high level of genetic variations within and between the parasite populations. Population-specific genetic studies to survey for genes under positive or balancing selection resulting from drug pressure or host immunity will allow for refinement of interventions.MethodsWe performed a pooled sequencing (pool-seq) of the genomes of 100 Plasmodium falciparum isolates from Nigeria. We explored allele-frequency based neutrality test (Tajima’s D) and integrated haplotype score (iHS) to identify genes under selection.ResultsFourteen shared iHS regions that had at least 2 SNPs with a score > 2.5 were identified. These regions code for genes that were likely to have been under strong directional selection. Two of these genes were the chloroquine resistance transporter (CRT) on chromosome 7 and the multidrug resistance 1 (MDR1) on chromosome 5. There was a weak signature of selection in the dihydrofolate reductase (DHFR) gene on chromosome 4 and MDR5 genes on chromosome 13, with only 2 and 3 SNPs respectively identified within the iHS window. We observed strong selection pressure attributable to continued chloroquine and sulfadoxine-pyrimethamine use despite their official proscription for the treatment of uncomplicated malaria. There was also a major selective sweep on chromosome 6 which had 32 SNPs within the shared iHS region. Tajima’s D of circumsporozoite protein (CSP), erythrocyte-binding antigen (EBA-175), merozoite surface proteins - MSP3 and MSP7, merozoite surface protein duffy binding-like (MSPDBL2) and serine repeat antigen (SERA-5) were 1.38, 1.29, 0.73, 0.84 and 0.21, respectively.ConclusionWe have demonstrated the use of pool-seq to understand genomic patterns of selection and variability in P. falciparum from Nigeria, which bears the highest burden of infections. This investigation identified known genomic signatures of selection from drug pressure and host immunity. This is evidence that P. falciparum populations explore common adaptive strategies that can be targeted for the development of new interventions.

Plasmodium falciparum erythrocyte-binding antigen 175 triggers a biophysical change in the red blood cell that facilitates invasion

Invasion of the red blood cell (RBC) by the Plasmodium parasite defines the start of malaria disease pathogenesis. To date, experimental investigations into invasion have focused predominantly on the role of parasite adhesins or signaling pathways and the identity of binding receptors on the red cell surface. A potential role for signaling pathways within the erythrocyte, which might alter red cell biophysical properties to facilitate invasion, has largely been ignored. The parasite erythrocyte-binding antigen 175 (EBA175), a protein required for entry in most parasite strains, plays a key role by binding to glycophorin A (GPA) on the red cell surface, although the function of this binding interaction is unknown. Here, using real-time deformability cytometry and flicker spectroscopy to define biophysical properties of the erythrocyte, we show that EBA175 binding to GPA leads to an increase in the cytoskeletal tension of the red cell and a reduction in the bending modulus of the cell's membrane. We isolate the changes in the cytoskeleton and membrane and show that reduction in the bending modulus is directly correlated with parasite invasion efficiency. These data strongly imply that the malaria parasite primes the erythrocyte surface through its binding antigens, altering the biophysical nature of the target cell and thus reducing a critical energy barrier to invasion. This finding would constitute a major change in our concept of malaria parasite invasion, suggesting it is, in fact, a balance between parasite and host cell physical forces working together to facilitate entry.

Plasmodium falciparum ligand binding to erythrocytes induce alterations in deformability essential for invasion.

The most lethal form of malaria in humans is caused by Plasmodium falciparum. These parasites invade erythrocytes, a complex process involving multiple ligand-receptor interactions. The parasite makes initial contact with the erythrocyte followed by dramatic deformations linked to the function of the Erythrocyte binding antigen family and P. falciparum reticulocyte binding-like families. We show EBA-175 mediates substantial changes in the deformability of erythrocytes by binding to glycophorin A and activating a phosphorylation cascade that includes erythrocyte cytoskeletal proteins resulting in changes in the viscoelastic properties of the host cell. TRPM7 kinase inhibitors FTY720 and waixenicin A block the changes in the deformability of erythrocytes and inhibit merozoite invasion by directly inhibiting the phosphorylation cascade. Therefore, binding of P. falciparum parasites to the erythrocyte directly activate a signaling pathway through a phosphorylation cascade and this alters the viscoelastic properties of the host membrane conditioning it for successful invasion.

Evolution of Fseg/Cseg dimorphism in region III of the Plasmodium falciparum eba-175 gene

The 175-kDa erythrocyte binding antigen (EBA-175) of the malaria parasite Plasmodium falciparum is important for its invasion into human erythrocytes. The primary structure of eba-175 is divided into seven regions, namely I to VII. Region III contains highly divergent dimorphic segments, termed Fseg and Cseg. The allele frequencies of segmental dimorphism within a P. falciparum population have been extensively examined; however, the molecular evolution of segmental dimorphism is not well understood. A comprehensive comparison of nucleotide sequences among 32 P. falciparum eba-175 alleles identified in our previous study, two Plasmodium reichenowi, and one P. gaboni orthologous alleles obtained from the GenBank database was conducted to uncover the origin and evolutionary processes of segmental dimorphism in P. falciparum eba-175. In the eba-175 nucleotide sequence derived from a P. reichenowi CDC strain, both Fseg and Cseg were found in region III, which implies that the original eba-175 gene had both segments, and deletions of F- and C-segments generated Cseg and Fseg alleles, respectively. We also confirmed the presence of allele with Fseg and Cseg in another P. reichenowi strain (SY57), by re-mapping short reads obtained from the GenBank database. On the other hand, the segmental sequence of eba-175 ortholog in P. gaboni was quite diverged from those of the other species, suggesting that the original eba-175 dimorphism of P. falciparum can be traced back to the stem linage of P. falciparum and P. reichenowi. Our findings suggest that Fseg and Cseg alleles are derived from a single eba-175 allele containing both segments in the ancestral population of P. falciparum and P. reichenowi, and that the allelic dimorphism of eba-175 was shaped by the independent emergence of similar dimorphic lineage in different species that has never been observed in any evolutionary mode of allelic dimorphism at other loci in malaria genomes.

Trafficking of Plasmodium Falciparum Proteins: A Novel Anti-Malaria Drug Target?

Malaria continues to be a global health problem and in 2015 the World Health Organisation estimated that there were ~214 million cases of the disease and ~438 000 deaths. Despite a marked reduction in mortality and morbidity over the last 15 years, these hard fought gains are threatened by resistance of the Anopheles mosquito vector to insecticides and resistance of the malaria parasite to artemisinin, currently the main frontline drug used in treatment of the disease. There is no effective vaccine and thus new drug targets are needed to fuel the development of compounds that kill parasites. Plasmodium falciparum is the most lethal of the malaria parasites, but our knowledge of the fundamental biology of this parasite is still incomplete and ~60% of the proteome is uncharacterised.This study aims to identify unique proteins/pathways involved in internal trafficking of parasite proteins. Clinical symptoms of malaria are caused by the intra-erythrocytic phase of the parasite life cycle and entry into the erythrocyte is accomplished by several specialised invasion proteins, which are stored in unique apical secretory organelles known as micronemes and rhoptries. Very little is known about the trafficking signals and transport mechanisms of invasion proteins to the apical organelles. Three micronemal proteins, apical membrane antigen-1 (PfAMA-1), subtilisin 2 (PfSUB2) and Erythrocyte Binding Antigen 181 (PfEBA-181) were investigated. Bioinformatic analysis revealed that the proteins contain N-terminal signal peptides, which direct the newly synthesised invasion proteins to the endoplasmic reticulum (ER). Transmembrane domains and cytoplasmic tails are located in the C-terminal ends and various functional domains are present in the central sections.To identify the domains responsible for targeting the micronemes, selected domains were amplified by PCR or RT-PCR from P. falciparum genomic DNA or RNA and used to create 7 mini-genes by overlap extension PCR. All products contained the N-terminal signal peptide that is required for transport through the ER. A pARL-mCherry plasmid (kindly donated by Jude Przyborski) was modified to replace the constitutive Pfcrt promoter with a Pfama-1 promoter that is only active during the schizont stage of parasite development when micronemes are formed. The plasmid contains a positive selection cassette that codes for human dihydrofolate reductase, which is resistant to WR99210 drug. The mini-genes were cloned into the modified plasmid and these constructs were used to transfect P. falciparum parasites by electroporation. Transgenic parasites were selected by WR99210 drug pressure and the expression of red fluorescent mCherry-tagged chimeric proteins was visualised in live parasites. Co-localisation studies were performed with green fluorescent PfEBA-175, a microneme marker, to assess if the transgenic mini-proteins reached their destination. In line with other EBAs, PfEBA-181 required the cysteine-rich ectodomain for correct localisation, whereas the prodomain of PfAMA-1 and transmembrane domain of PfSUB2 were essential for microneme targeting.Since these 3 invasion proteins all require different domains for transport to the micronemes, the possibility of a protein escorter was explored. The PfAMA-1 prodomain was expressed as a recombinant histidine tagged "bait" protein and immobilised onto Nickel-coated beads, which were exposed to a P. falciparum phage display library for 4 rounds of biopanning. Phage that bound to the bait were isolated and the parasite inserts were sequenced. Comparison of the sequences to the Plasmodium database revealed 2 binding partners: a putative chaperone binding protein and a putative P. falciparum formin 2. The human orthologue of formin has several functions in the actin cytoskeleton, including intracellular vesicle transport along the actin fibres. These binding partners are currently being expressed as GST-tagged recombinant proteins, which will be used in in vitro binding assays to verify their interaction with the prodomain of PfAMA-1. To ascertain if formin 2 and chaperone binding protein are common transporters for micronemal invasion proteins, their interaction with the targeting domains of PfSUB2 and PfEBA-181 will be investigated. Since humans don't have apical organelles, these trafficking mechanisms are unique to P. falciparum and may be exploited as novel anti-malaria drug targets. DisclosuresNo relevant conflicts of interest to declare.

Safety and Immunogenicity of EBA-175 RII-NG Malaria Vaccine Administered Intramuscularly in Semi-Immune Adults: A Phase 1, Double-Blinded Placebo Controlled Dosage Escalation Study.

The erythrocyte binding antigen region II (EBA-175 RII) is a Plasmodium falciparum ligand that mediates erythrocyte invasion and is considered an important malaria vaccine candidate. A phase Ia trial in malaria naïve adults living in the United States found the recombinant non-glycosylated vaccine antigen, EBA-175 RII-NG adjuvanted with aluminium phosphate to be safe, immunogenic and capable of inducing biologically active antibodies that can inhibit parasite growth in vitro. The aim of the current study was to assess the safety and immunogenicity of this vaccine in malaria exposed semi-immune healthy adults living in a malaria endemic country, Ghana. In this double-blinded, placebo controlled, dose escalation phase I trial, eighteen subjects per group received ascending dose concentrations (5 μg, 20 μg or 80 μg) of the vaccine intramuscularly at 0, 1 and 6 months, while 6 subjects received placebo (normal saline). The primary end point was the number of subjects experiencing Grade 3 systemic or local adverse events within 14 days post-vaccination. Serious adverse events were assessed throughout the study period. Blood samples for immunological analyses were collected at days 0, 14, 28, 42, 180 and 194. A total of 52 subjects received three doses of the vaccine in the respective groups. No serious adverse events were reported. The majority of all adverse events reported were mild to moderate in severity, with local pain and tenderness being the most common. All adverse events, irrespective of severity, resolved without any sequelae. Subjects who received any of the EBA-175 RII-NG doses had high immunoglobulin G levels which moderately inhibited P. falciparum growth in vitro, compared to those in the placebo group. In conclusion, the EBA-175 RII-NG vaccine was safe, well tolerated and immunogenic in malaria semi-immune Ghanaian adults. Its further development is recommended.Trial registrationClinicalTrials.gov. Identifier: NCT01026246

Baculovirus-expressed Plasmodium reichenowi EBA-140 merozoite ligand is host specific

Plasmodium reichenowi, an ape malaria parasite is morphologically identical and genetically similar to Plasmodium falciparum, infects chimpanzees but not humans. Genomic studies revealed that all primate malaria parasites belong to Laverania subgenus. Laverania parasites exhibit strict host specificity, but the molecular mechanisms underlying these host restrictions remain unexplained.Plasmodium merozoites express multiple binding ligands that recognize specific receptors on erythrocytes, including micronemal proteins belonging to P. falciparum EBL family. It was shown that erythrocyte binding antigen-175 (EBA-175), erythrocyte binding ligand-1 (EBL-1), erythrocyte binding antigen-140 (EBA-140) recognize erythrocyte surface sialoglycoproteins - glycophorins A, B, C, respectively. EBA-140 merozoite ligand hijacks glycophorin C (GPC), a minor erythrocyte sialoglycoprotein, to invade the erythrocyte through an alternative invasion pathway.A homolog of P. falciparum EBA-140 protein was identified in P. reichenowi. The amino acid sequences of both EBA-140 ligands are very similar, especially in the conservative erythrocyte binding region (Region II). It has been suggested that evolutionary changes in the sequence of EBL proteins may be associated with Plasmodium host restriction.In this study we obtained, for the first time, the recombinant P. reichenowi EBA-140 ligand Region II using baculovirus expression vector system. We show that the ape EBA-140 Region II is host specific and binds to chimpanzee erythrocytes in the dose and sialic acid dependent manner. Further identification of the erythrocyte receptor for this ape ligand is of great interests, since it may reveal the molecular basis of host restriction of both P. reichenowi and its deadliest human counterpart, P. falciparum.

Age-dependent carriage of alleles and haplotypes of Plasmodium falciparum sera5, eba-175, and csp in a region of intense malaria transmission in Uganda

Background: The development of malaria vaccines is constrained by genetic polymorphisms exhibited by Plasmodium falciparum antigens. We investigated the age-dependent distribution of alleles or haplotypes of three P. falciparum malaria vaccine candidates, circumsporozoite protein (csp), erythrocyte binding antigen 175 (eba-175) and serine repeat antigen 5 (sera5) in a region of intense malaria transmission in Uganda. Methods & Materials: A cross sectional study was carried out between August and November 2009. Blood samples were collected after informed consent from 250 individuals below 5 years, 5-10 years and above 10 years olds. P. falciparum DNA was extracted from all samples. Alleles of sera5 and eba-175 were determined by polymerase chain reaction (PCR) amplification followed by resolution of PCR products by agarose gel electrophoresis and allele calling using photographs of ethidium bromide-stained gels. Haplotypes of CSP were identified by sequencing 63 PCR products and using P.falciparum 7G8 strain sequence as a reference. Results: Both eba-175 FCR3 (48/178) and CAMP (16/178) alleles were observed with the FCR3 (24/67) allele being predominant among children aged below 5 years old while the CAMP (12/67) allele was predominant among older individuals. Both sera5 alleles ORI (6/204) and ORII (103/204) were observed in the population but ORII was more prevalent. SERA5 ORII allele was significantly associated with age (P values < 0.0001), parasite density (P value < 0.0001) and clinical outcomes (P value = 0.018). There was marked CSP diversity in the Th2/Th3 region. Out of 63 sequences, 16 conformed to the reference strain and one (1/16) was similar with a West African haplotype and the majority (14/16) of the haplotypes were unique to this study region. Conclusion: There was an age-dependent distribution of CSP haplotypes with more haplotypes being harbored by < 5-year olds, (10/16) compared to adults (2/16). Interestingly, the CSP haplotype corresponding to 3D7 whose prototypical sequence is identical to the sequence of the leading malaria vaccine candidate RTS, S was not observed. Our data suggest that eba-175 FCR3 allele, sera5 ORII allele, and CSP haplotypes are targets of host immunity and under immune selection pressure in Apac District.