P. Vivax Duffy Binding Protein Research Articles

Complement receptor 1 is the human erythrocyte receptor for Plasmodium vivax erythrocyte binding protein

The discovery that Africans were resistant to infection by Plasmodium vivax (P. vivax) led to the conclusion that P. vivax invasion relied on the P. vivax Duffy Binding Protein (PvDBP) interacting with the Duffy Antigen Receptor for Chemokines (DARC) expressed on erythrocytes. However, the recent reporting of P. vivax infections in DARC-negative Africans suggests that the parasite might use an alternate invasion pathway to infect DARC-negative reticulocytes. To identify the parasite ligands and erythrocyte receptors that enable P. vivax invasion of both DARC-positive and -negative erythrocytes, we expressed region II containing the Duffy Binding-Like (DBL) domain of P. vivax erythrocyte binding protein (PvEBP-RII) and verified that the DBL domain binds to both DARC-positive and -negative erythrocytes. Furthermore, an AVidity-based EXtracelluar Interaction Screening (AVEXIS) was used to identify the receptor for PvEBP among over 750 human cell surface receptor proteins, and this approach identified only Complement Receptor 1 (CR1, CD35, or C3b/C4b receptor) as a PvEBP receptor. CR1 is a well-known receptor for P. falciparum Reticulocyte binding protein Homology 4 (PfRh4) and is present on the surfaces of both reticulocytes and normocytes, but its expression decreases as erythrocytes age. Indeed, PvEBP-RII bound to a subpopulation of both reticulocytes and normocytes, and this binding was blocked by the addition of soluble CR1 recombinant protein, indicating that CR1 is the receptor of PvEBP. In addition, we found that the Long Homology Repeat A (LHR-A) subdomain of CR1 is the only subdomain responsible for mediating the interaction with PvEBP-RII.

Unveiling P. vivax invasion pathways in Duffy-negative individuals

Vivax malaria has long been thought to be absent from sub-Saharan Africa owing to the high proportion of individuals lacking the Duffy antigen receptor for chemokines (DARC) in their erythrocytes. The interaction between P.vivax Duffy-binding protein (PvDBP) and DARC is assumed to be the main pathway used by merozoites to invade reticulocytes. However, the increasing number of reports of vivax malaria cases in genotypically Duffy-negative (DN) individuals has raised questions regarding the P.vivax invasion pathway(s). Here, we show that a subset of DN erythroblasts transiently express DARC during terminal erythroid differentiation and that P.vivax merozoites, irrespective of their origin, can invade DARC+ DN erythroblasts. These findings reveal that a large number of DN individuals may represent a silent reservoir of deep P.vivax infections at the sites of active erythropoiesis with low or no parasitemia, and it may represent an underestimated biological problem with potential clinical consequences in sub-Saharan Africa.

Plasmodium vivax merozoite-specific thrombospondin-related anonymous protein (PvMTRAP) interacts with human CD36, suggesting a novel ligand–receptor interaction for reticulocyte invasion

BackgroundThe Plasmodium vivax merozoite restrictively invades immature erythrocytes, suggesting that its ligand(s) might interact with corresponding receptor(s) that are selectively abundant on reticulocytes to complete the invasion. Finding the ligand‒receptor interaction involved in P. vivax invasion is critical to vivax malaria management; nevertheless, it remains to be unraveled.MethodsA library of reticulocyte receptors and P. vivax ligands were expressed by a HEK293E mammalian cell expression system and were then used to screen the interaction using enzyme-linked immunosorbent assay (ELISA). A flow cytometry-based erythrocyte binding assay and bio-layer interferometry experiment were further utilized to cellularly and quantitatively identify the ligand‒receptor interaction, respectively.ResultsPlasmodium vivax merozoite-specific thrombospondin-related anonymous protein (PvMTRAP) was found to interact with human CD36 using systematic screening. This interaction was specific at a molecular level from in vitro analysis and comparable to that of P. vivax Duffy binding protein (PvDBP) and Duffy antigen receptor for chemokines (DARC) (KD: 37.0 ± 1.4 nM and 7.7 ± 0.5 nM, respectively). Flow cytometry indicated that PvMTRAP preferentially binds to reticulocytes, on which CD36 is selectively present.ConclusionsHuman CD36 is selectively abundant on reticulocytes and is able to interact specifically with PvMTRAP, suggesting that it may function as a ligand and receptor during the invasion of reticulocytes by P. vivax.Graphical

The Darc Side of Vivax Malaria in Africa: Unveiling Invasion Pathways into Duffy-Negative Erythroblasts

Background & Objectives: Plasmodium vivax malaria was long thought to be absent from sub-Saharan Africa owing to the high prevalence of people lacking the Duffy antigen receptor for chemokines (DARC) on their erythrocytes. The interaction between P. vivax Duffy binding protein (PvDBP) and DARC is assumed to be the main mechanism used by P. vivax merozoites to invade human erythrocytes. However, the increasing numbers of vivax malaria cases in Duffy-negative African individuals has raised questions about alternative P. vivax invasion pathway(s) other than PvDBP-DARC interaction. Since P. vivax has a tropism for CD71+ immature reticulocytes and the hematopoietic niches of the bone marrow may be a hidden reservoir of P. vivax parasites, we hypothesized that P. vivax merozoites may be able to invade erythroblasts derived from Duffy-positive (DP) and Duffy-negative (DN) individuals. Therefore, our objectives were to (i) investigate the expression of DARC during DP and DN erythropoiesis and (ii) examine the infection of DP and DN erythroblasts with P. vivax merozoites in vitro to study and identify host receptors used by P. vivax to invade erythroblasts. Methods: We performed in vitro erythropoiesis assays (two-phase culture) of genotypically DN and DP erythroid progenitors. Erythroid differentiation, CD71 and DARC expression profiles were monitored using flow cytometry, microscopy, and western blot over time during phase 2. In vitro invasion assays were conducted using DP and DN erythroblasts to investigate whether genotypically DN erythroblasts could be invaded by P. vivax. At D7 of phase 2, we thawed and cultured parasite isolates obtained from two Malagasy and seven Ethiopian patients infected with P. vivax. After approximately 24-30 hours of in vitro maturation (at D8 of phase 2), each P. vivax parasite culture was placed in co-culture with either DP or DN erythroblasts for 24-48 hours. We examined the infection of DP and DN erythroblasts by P. vivax parasites by light and confocal microscopy. Results: Using in vitro erythropoiesis, we show that both genotypically DP and DN erythroblasts have similar kinetics of terminal erythroid differentiation. Moreover, we found similar CD71 expression profiles in both DP and DN erythroblasts. Interestingly, we observed that a subset of DN erythroblasts transiently expressed DARC during erythropoiesis. This was confirmed using both flow cytometry and western blot analysis. In vitro invasion assays conducted on both DP and DN erythroblasts using parasite isolates, showed that P. vivax merozoites were able to invade both DP and DN erythroblasts (Figure 1). We found that all the DP and DN erythroblasts that were infected with P. vivax were CD71+ and DARC+. These results confirm that P. vivax can invade DP and DN erythroblasts expressing both DARC and CD71, irrespective of the origin of the isolates. Conclusion: This study provides the first biological evidence for the ability of P. vivax to invade genotypically DN erythroblasts and highlights that African populations may represent a silent reservoir of P. vivax infections, with major implications for the control and elimination of vivax malaria in sub-Saharan Africa.

A conserved epitope in VAR2CSA is targeted by a cross-reactive antibody originating from Plasmodium vivax Duffy binding protein.

During Plasmodium falciparum infection in pregnancy, VAR2CSA is expressed on the surface of infected erythrocytes (IEs) and mediates their sequestration in the placenta. As a result, antibodies to VAR2CSA are largely restricted to women who were infected during pregnancy. However, we discovered that VAR2CSA antibodies can also be elicited by P. vivax Duffy binding protein (PvDBP). We proposed that infection with P. vivax in non-pregnant individuals can generate antibodies that cross-react with VAR2CSA. To better understand the specificity of these antibodies, we took advantage of a mouse monoclonal antibody (3D10) raised against PvDBP that cross-reacts with VAR2CSA and identified the epitopes targeted by this antibody. We screened two peptide arrays that span the ectodomain of VAR2CSA from the FCR3 and NF54 alleles. Based on the top epitope recognized by 3D10, we designed a 34-amino acid synthetic peptide, which we call CRP1, that maps to a highly conserved region in DBL3X. Specific lysine residues are critical for 3D10 recognition, and these same amino acids are within a previously defined chondroitin sulfate A (CSA) binding site in DBL3X. We showed by isothermal titration calorimetry that the CRP1 peptide can bind directly to CSA, and antibodies to CRP1 raised in rats significantly blocked the binding of IEs to CSA in vitro. In our Colombian cohorts of pregnant and non-pregnant individuals, at least 45% were seroreactive to CRP1. Antibody reactivities to CRP1 and the 3D10 natural epitope in PvDBP region II, subdomain 1 (SD1), were strongly correlated in both cohorts. These findings suggest that antibodies arising from PvDBP may cross-react with VAR2CSA through the epitope in CRP1 and that CRP1 could be a potential vaccine candidate to target a distinct CSA binding site in VAR2CSA.

APC-Targeted DNA Vaccination Against Reticulocyte-Binding Protein Homolog 5 Induces Plasmodium falciparum-Specific Neutralizing Antibodies and T Cell Responses.

Targeted delivery of antigen to antigen presenting cells (APCs) is an efficient way to induce robust antigen-specific immune responses. Here, we present a novel DNA vaccine that targets the Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5), a leading blood-stage antigen of the human malaria pathogen, to APCs. The vaccine is designed as bivalent homodimers where each chain is composed of an amino-terminal single chain fragment variable (scFv) targeting unit specific for major histocompatibility complex class II (MHCII) expressed on APCs, and a carboxyl-terminal antigenic unit genetically linked by the dimerization unit. This vaccine format, named “Vaccibody”, has previously been successfully applied for antigens from other infectious diseases including influenza and HIV, as well as for tumor antigens. Recently, the crystal structure and key functional antibody epitopes for the truncated version of PfRH5 (PfRH5ΔNL) were characterized, suggesting PfRH5ΔNL to be a promising candidate for next-generation PfRH5 vaccine design. In this study, we explored the APC-targeting strategy for a PfRH5ΔNL-containing DNA vaccine. BALB/c mice immunized with the targeted vaccine induced higher PfRH5-specific IgG1 antibody responses than those vaccinated with a non-targeted vaccine or antigen alone. The APC-targeted vaccine also efficiently induced rapid IFN-γ and IL-4 T cell responses. Furthermore, the vaccine-induced PfRH5-specific IgG showed inhibition of growth of the P. falciparum 3D7 clone parasite in vitro. Finally, sera obtained after vaccination with this targeted vaccine competed for the same epitopes as PfRH5-specific mAbs from vaccinated humans. Robust humoral responses were also induced by a similar P. vivax Duffy-binding protein (PvDBP)-containing targeted DNA vaccine. Our data highlight a novel targeted vaccine platform for the development of vaccines against blood-stage malaria.

The enigmatic mechanisms by which Plasmodium vivax infects Duffy-negative individuals.

The absence of the Duffy protein at the surface of erythrocytes was considered for decades to confer full protection against Plasmodium vivax as this blood group is the receptor for the key parasite ligand P. vivax Duffy binding protein (PvDBP). However, it is now clear that the parasite is able to break through this protection and induce clinical malaria in Duffy-negative people, although the underlying mechanisms are still not understood. Here, we briefly review the evidence of Duffy-negative infections by P. vivax and summarize the current hypothesis at the basis of this invasion process. We discuss those in the perspective of malaria-elimination challenges, notably in African countries.

Rapid and iterative genome editing in the malaria parasite Plasmodium knowlesi provides new tools for P. vivax research.

Tackling relapsing Plasmodium vivax and zoonotic Plasmodium knowlesi infections is critical to reducing malaria incidence and mortality worldwide. Understanding the biology of these important and related parasites was previously constrained by the lack of robust molecular and genetic approaches. Here, we establish CRISPR-Cas9 genome editing in a culture-adapted P. knowlesi strain and define parameters for optimal homology-driven repair. We establish a scalable protocol for the production of repair templates by PCR and demonstrate the flexibility of the system by tagging proteins with distinct cellular localisations. Using iterative rounds of genome-editing we generate a transgenic line expressing P. vivax Duffy binding protein (PvDBP), a lead vaccine candidate. We demonstrate that PvDBP plays no role in reticulocyte restriction but can alter the macaque/human host cell tropism of P. knowlesi. Critically, antibodies raised against the P. vivax antigen potently inhibit proliferation of this strain, providing an invaluable tool to support vaccine development.

Structural basis for inhibition of Plasmodium vivax invasion by a broadly neutralizing vaccine-induced human antibody.

SummaryThe most widespread form of malaria is caused by Plasmodium vivax. To replicate, this parasite must invade immature red blood cells, through a process which requires interaction of the Plasmodium vivax Duffy binding protein, PvDBP with its human receptor, the Duffy antigen receptor for chemokines, DARC. Naturally acquired antibodies that inhibit this interaction associate with clinical immunity, suggesting PvDBP as a leading candidate for inclusion in a vaccine to prevent malaria due to Plasmodium vivax. Here, we isolated a panel of monoclonal antibodies from human volunteers immunised in a clinical vaccine trial of PvDBP. We screened their ability to prevent PvDBP from binding to DARC, and their capacity to block red blood cell invasion by a transgenic Plasmodium knowlesi parasite genetically modified to express PvDBP and to prevent reticulocyte invasion by multiple clinical isolates of Plasmodium vivax. This identified a broadly neutralising human monoclonal antibody which inhibited invasion of all tested strains of Plasmodium vivax. Finally, we determined the structure of a complex of this antibody bound to PvDBP, indicating the molecular basis for inhibition. These findings will guide future vaccine design strategies and open up possibilities for testing the prophylactic use of such an antibody.

Cross-Species Immune Recognition Between Plasmodium vivax Duffy Binding Protein Antibodies and the Plasmodium falciparum Surface Antigen VAR2CSA.

In pregnancy, Plasmodium falciparum parasites express the surface antigen VAR2CSA, which mediates adherence of red blood cells to chondroitin sulfate A (CSA) in the placenta. VAR2CSA antibodies are generally acquired during infection in pregnancy and are associated with protection from placental malaria. We observed previously that men and children in Colombia also had antibodies to VAR2CSA, but the origin of these antibodies was unknown. Here, we tested whether infection with Plasmodium vivax is an alternative mechanism of acquisition of VAR2CSA antibodies. We analyzed sera from nonpregnant Colombians and Brazilians exposed to P. vivax and monoclonal antibodies raised against P. vivax Duffy binding protein (PvDBP). Cross-reactivity to VAR2CSA was characterized by enzyme-linked immunosorbent assay, immunofluorescence assay, and flow cytometry, and antibodies were tested for inhibition of parasite binding to CSA. Over 50% of individuals had antibodies that recognized VAR2CSA. Affinity-purified PvDBP human antibodies and a PvDBP monoclonal antibody recognized VAR2CSA, showing that PvDBP can give rise to cross-reactive antibodies. Importantly, the monoclonal antibody inhibited parasite binding to CSA, which is the primary in vitro correlate of protection from placental malaria. These data suggest that PvDBP induces antibodies that functionally recognize VAR2CSA, revealing a novel mechanism of cross-species immune recognition to falciparum malaria.

Targeting a Reticulocyte Binding Protein and Duffy Binding Protein to Inhibit Reticulocyte Invasion by Plasmodium vivax

Plasmodium vivax merozoite invasion is restricted to Duffy positive reticulocytes. Merozoite interaction with the Duffy antigen is mediated by the P. vivax Duffy binding protein (PvDBP). The receptor-binding domain of PvDBP maps to an N-terminal cysteine-rich region referred to as region II (PvDBPII). In addition, a family of P. vivax reticulocyte binding proteins (PvRBPs) mediates interactions with reticulocyte receptors. The receptor binding domain of P. vivax reticulocyte binding protein 1a (PvRBP1a) maps to a 30 kD region (PvRBP1a30). Antibodies raised against recombinant PvRBP1a30 and PvDBPII recognize the native P. vivax antigens and inhibit their binding to host receptors. Rabbit IgG purified from sera raised against PvRBP1a30 and PvDBPII were tested individually and in combination for inhibition of reticulocyte invasion by P. vivax field isolates. While anti-PvDBPII rabbit IgG inhibits invasion, anti-PvRBP1a30 rabbit IgG does not show significant invasion inhibitory activity. Combining antibodies against PvDBPII and PvRBP1a30 also does not increase invasion inhibitory activity. These studies suggest that although PvRBP1a mediates reticulocyte invasion by P. vivax merozoites, it may not be useful to include PvRBP1a30 in a blood stage vaccine for P. vivax malaria. In contrast, these studies validate PvDBPII as a promising blood stage vaccine candidate for P. vivax malaria.

Genetic polymorphism of Plasmodium vivax Duffy Binding Protein in malarious areas in southeastern of Iran.

Plasmodium vivax parasite causes the largest number of malaria infection in some malarious areas of the world including Iran. Considering transfer and genetic dynamics of the parasite population in a specific area can help us to predict the spread of the infection either emergence of new cases or drug resistance in the context of elimination program in the malarious areas. Study on the genetic diversity of common alleles in a given geographical area, for vaccine and immune level studies can be important. The purpose of this study was to know the status of P. vivax Duffy Binding Protein (PvDBP) polymorphism in patients infected with the parasite in malaria endemic southeastern Iran. The fragment of gene corresponding to PvDBP of thirty P. vivax malaria infected individuals was amplified. A 1176bp band related to this fragment was purified and PCR-RFLP method was employed using enzymatic digestion with PstI and RsaI restriction enzymes. Ten percent of samples were sent for sequencing. PCR-RFLP showed that 99.7% of the samples were cut as the same together, either the PstI enzyme or the enzyme of RsaI. In each case, only 2 isolates were unlike others. Findings revealed that there is at least 96% identity among isolates in the nucleotide level. Amino acid pattern of PvDBP in Iranian isolates showed little discrepancies with those PvDBP genes that have been recorded in GenBank. Sequencing of PvDBP isolates of Iranian P. vivax infected patients showed low level of genetic polymorphism among them. Results of this study can prepare valuable information for malaria policy makers to intend them in their malaria control program.

Independent Origin and Global Distribution of Distinct Plasmodium vivax Duffy Binding Protein Gene Duplications.

BackgroundPlasmodium vivax causes the majority of malaria episodes outside Africa, but remains a relatively understudied pathogen. The pathology of P. vivax infection depends critically on the parasite’s ability to recognize and invade human erythrocytes. This invasion process involves an interaction between P. vivax Duffy Binding Protein (PvDBP) in merozoites and the Duffy antigen receptor for chemokines (DARC) on the erythrocyte surface. Whole-genome sequencing of clinical isolates recently established that some P. vivax genomes contain two copies of the PvDBP gene. The frequency of this duplication is particularly high in Madagascar, where there is also evidence for P. vivax infection in DARC-negative individuals. The functional significance and global prevalence of this duplication, and whether there are other copy number variations at the PvDBP locus, is unknown.Methodology/Principal FindingsUsing whole-genome sequencing and PCR to study the PvDBP locus in P. vivax clinical isolates, we found that PvDBP duplication is widespread in Cambodia. The boundaries of the Cambodian PvDBP duplication differ from those previously identified in Madagascar, meaning that current molecular assays were unable to detect it. The Cambodian PvDBP duplication did not associate with parasite density or DARC genotype, and ranged in prevalence from 20% to 38% over four annual transmission seasons in Cambodia. This duplication was also present in P. vivax isolates from Brazil and Ethiopia, but not India.Conclusions/SignificancePvDBP duplications are much more widespread and complex than previously thought, and at least two distinct duplications are circulating globally. The same duplication boundaries were identified in parasites from three continents, and were found at high prevalence in human populations where DARC-negativity is essentially absent. It is therefore unlikely that PvDBP duplication is associated with infection of DARC-negative individuals, but functional tests will be required to confirm this hypothesis.

Production of recombinant PvDBPII, receptor binding domain of Plasmodium vivax Duffy binding protein, and evaluation of immunogenicity to identify an adjuvant formulation for vaccine development

Plasmodium vivax is dependent on interaction with the Duffy antigen receptor for chemokines (DARC) for invasion of human erythrocytes. The P. vivax Duffy binding protein (PvDBP) mediates interaction of P. vivax merozoites with DARC. The DARC receptor-binding domain lies in a conserved N-terminal cysteine-rich region of PvDBP referred to as region II (PvDBPII). PvDBPII is an attractive vaccine candidate since antibodies raised against PvDBPII block erythrocyte invasion by P. vivax. Here, we describe methods to produce recombinant PvDBPII in its correctly folded conformation. A synthetic gene optimized for expression of PvDBPII in Escherichia coli and fed batch fermentation process based on exponential feeding strategy was used to achieve high levels of expression of recombinant PvDBPII. Recombinant PvDBPII was isolated from inclusion bodies, refolded by rapid dilution and purified by ion exchange chromatography. Purified recombinant PvDBPII was characterized for identity, purity and functional activity using standardized release assays. Recombinant PvDBPII formulated with various human compatible adjuvants including glycosylpyranosyl lipid A-stable emulsion (GLA-SE) and alhydrogel was used for immunogenicity studies in small animals to downselect a suitable formulation for clinical development. Sera collected from immunized animals were tested for recognition of PvDBPII and inhibition of PvDBPII-DARC binding. GLA-SE formulations of PvDBPII yielded higher ELISA and binding inhibition titres compared to PvDBPII formulated with alhydrogel. These data support further development of a recombinant vaccine for P. vivax based on PvDBPII formulated with GLA-SE.

Whole Genome Sequencing of Field Isolates Reveals a Common Duplication of the Duffy Binding Protein Gene in Malagasy Plasmodium vivax Strains

Background Plasmodium vivax is the most prevalent human malaria parasite, causing serious public health problems in malaria-endemic countries. Until recently the Duffy-negative blood group phenotype was considered to confer resistance to vivax malaria for most African ethnicities. We and others have reported that P. vivax strains in African countries from Madagascar to Mauritania display capacity to cause clinical vivax malaria in Duffy-negative people. New insights must now explain Duffy-independent P. vivax invasion of human erythrocytes.Methods/Principal FindingsThrough recent whole genome sequencing we obtained ≥70× coverage of the P. vivax genome from five field-isolates, resulting in ≥93% of the Sal I reference sequenced at coverage greater than 20×. Combined with sequences from one additional Malagasy field isolate and from five monkey-adapted strains, we describe here identification of DNA sequence rearrangements in the P. vivax genome, including discovery of a duplication of the P. vivax Duffy binding protein (PvDBP) gene. A survey of Malagasy patients infected with P. vivax showed that the PvDBP duplication was present in numerous locations in Madagascar and found in over 50% of infected patients evaluated. Extended geographic surveys showed that the PvDBP duplication was detected frequently in vivax patients living in East Africa and in some residents of non-African P. vivax-endemic countries. Additionally, the PvDBP duplication was observed in travelers seeking treatment of vivax malaria upon returning home. PvDBP duplication prevalence was highest in west-central Madagascar sites where the highest frequencies of P. vivax-infected, Duffy-negative people were reported.Conclusions/SignificanceThe highly conserved nature of the sequence involved in the PvDBP duplication suggests that it has occurred in a recent evolutionary time frame. These data suggest that PvDBP, a merozoite surface protein involved in red cell adhesion is rapidly evolving, possibly in response to constraints imposed by erythrocyte Duffy negativity in some human populations.

Antibody Responses and Avidity of Naturally Acquired Anti-Plasmodium vivax Duffy Binding Protein (PvDBP) Antibodies in Individuals from an Area with Unstable Malaria Transmission

Plasmodium vivax remains an important cause of morbidity outside Africa, and no effective vaccine is available against this parasite. The P. vivax Duffy binding protein (PvDBP) is essential during merozoite invasion into erythrocytes, and it is a target for protective immunity against malaria. This investigation was designed to evaluate naturally acquired antibodies to two variant forms of PvDBP-II antigen (DBP-I and -VI) in malaria individuals (N = 85; median = 22 years) who were living in hypoendemic areas in Iran. The two PvDBP-II variants were expressed in Escherichia coli, and immunoglobulin G (IgG) isotype composition and avidity of naturally acquired antibodies to these antigens were measured using enzyme-linked immunosorbent assay (ELISA). Results showed that almost 32% of the studied individuals had positive antibody responses to the two PvDBP-II variants, and the prevalence of responders did not differ significantly (P > 0.05; χ(2) test). The IgG-positive samples exhibited 37.03% and 40.8% high-avidity antibodies for PvDBP-I and PvDBP-VI variants, respectively. Furthermore, high-avidity IgG1 antibody was found in 39.1% of positive sera for each examined variant antigen. The avidity of antibodies for both PvDBP variant antigens and the prevalence of responders with high- and intermediate-avidity IgG, IgG1, and IgG3 antibodies were similar in patients (P > 0.05; χ(2) test). Moreover, the prevalence of IgG antibody responses to the two variants significantly increased with exposure and host age. To sum up, the results provided additional data in our understanding of blood-stage immunity to PvDBP, supporting the rational development of an effective blood-stage vaccine based on this antigen.

Determination of the Molecular Basis for a Limited Dimorphism, N417K, in the Plasmodium vivax Duffy-Binding Protein

Invasion of human red blood cells by Plasmodium merozoites is vital for replication and survival of the parasite and, as such, is an attractive target for therapeutic intervention. Merozoite invasion is mediated by specific interactions between parasite ligands and host erythrocyte receptors. The P. vivax Duffy-binding protein (PvDBP) is heavily dependent on the interaction with the human Duffy blood group antigen/receptor for chemokines (DARC) for invasion. Region II of PvDBP contains many allelic polymorphisms likely to have arisen by host immune selection. Successful vaccine development necessitates a deeper understanding of the role of these polymorphisms in both parasite function and evasion of host immunity. A 3D structure of the homologous P. knowlesi DBP predicts that most variant residues are surface-exposed, including N417K, which is a dimorphic residue change that has previously been shown to be part of a linked haplotype that alters DBP sensitivity to inhibitory antibody. In natural isolates only two residues are found at this site, asparagine (N) and lysine (K). Site-directed mutagenesis of residue 417 was used to create a panel of 20 amino acid variants that were then examined for their binding phenotype and response to immune sera. Our results suggest that the observed dimorphism likely arose due to both structural requirements and immune selection pressure. To our knowledge, this is the first exhaustive examination of this kind of the role of a single amino acid residue in antigenic character and binding ability. Our results demonstrate that a single amino acid substitution can dramatically alter both the ability of the PvDBP to bind to human erythrocytes and its antigenic character.

Duffy blood group system and the malaria adaptation process in humans

Malaria is an acute infectious disease caused by the protozoa of the genus Plasmodium. The antigens of the Duffy Blood Group System, in addition to incompatibilities in transfusions and hemolytic disease of the newborn, are of great interest in medicine due to their association with the invasion of red blood cells by the parasite Plasmodium vivax. For invasions to occur an interaction between the parasites and antigens of the Duffy Blood Group System is necessary. In Caucasians six antigens are produced by the Duffy locus (Fya, Fyb, F3, F4, F5 and F6). It has been observed that Fy(a-b-) individuals are resistant to Plasmodium knowlesi and P. vivax infection, because the invasion requires at least one of these antigens. The P. vivax Duffy Binding Protein (PvDBP) is functionally important in the invasion process of these parasites in Duffy / DARC positive humans. The proteins or fractions may be considered, therefore, an important and potential inoculum to be used in immunization against malaria.

Plasmodium vivax Invasion of Human Erythrocytes Inhibited by Antibodies Directed against the Duffy Binding Protein

Background Plasmodium vivax invasion requires interaction between the human Duffy antigen on the surface of erythrocytes and the P. vivax Duffy binding protein (PvDBP) expressed by the parasite. Given that Duffy-negative individuals are resistant and that Duffy-negative heterozygotes show reduced susceptibility to blood-stage infection, we hypothesized that antibodies directed against region two of P. vivax Duffy binding protein (PvDBPII) would inhibit P. vivax invasion of human erythrocytes.Methods and FindingsUsing a recombinant region two of the P. vivax Duffy binding protein (rPvDBPII), polyclonal antibodies were generated from immunized rabbits and affinity purified from the pooled sera of 14 P. vivax–exposed Papua New Guineans. It was determined by ELISA and by flow cytometry, respectively, that both rabbit and human antibodies inhibited binding of rPvDBPII to the Duffy antigen N-terminal region and to Duffy-positive human erythrocytes. Additionally, using immunofluorescent microscopy, the antibodies were shown to attach to native PvDBP on the apical end of the P. vivax merozoite. In vitro invasion assays, using blood isolates from individuals in the Mae Sot district of Thailand, showed that addition of rabbit anti-PvDBPII Ab or serum (antibodies against, or serum containing antibodies against, region two of the Plasmodium vivax Duffy binding protein) (1:100) reduced the number of parasite invasions by up to 64%, while pooled PvDBPII antisera from P. vivax–exposed people reduced P. vivax invasion by up to 54%.ConclusionsThese results show, for what we believe to be the first time, that both rabbit and human antibodies directed against PvDBPII reduce invasion efficiency of wild P. vivax isolated from infected patients, and suggest that a PvDBP-based vaccine may reduce human blood-stage P. vivax infection.

Targeting the Plasmodium vivax Duffy-binding protein

Plasmodium vivax requires interaction with the Duffy antigen receptor for chemokines (DARC) to enable its invasion of human erythrocytes. Interaction with DARC is mediated by the P. vivax Duffy-binding protein (PvDBP) and is essential for junction formation, which is a key step in the invasion process. The receptor-binding domain of PvDBP maps to a conserved cysteine-rich region, referred to as region II (PvRII). Here, we review data on the interaction of PvRII with DARC and explore the potential of targeting this crucial receptor-ligand interaction to develop new intervention strategies against P. vivax.