Vivax Reticulocyte Binding Protein Research Articles

Geographical distribution and genetic diversity of Plasmodium vivax reticulocyte binding protein 1a correlates with patient antigenicity.

Plasmodium vivax is the most widespread cause of human malaria. Recent reports of drug resistant vivax malaria and the challenge of eradicating the dormant liver forms increase the importance of vaccine development against this relapsing disease. P. vivax reticulocyte binding protein 1a (PvRBP1a) is a potential vaccine candidate, which is involved in red cell tropism, a crucial step in the merozoite invasion of host reticulocytes. As part of the initial evaluation of the PvRBP1a vaccine candidate, we investigated its genetic diversity and antigenicity using geographically diverse clinical isolates. We analysed pvrbp1a genetic polymorphisms using 202 vivax clinical isolates from six countries. Pvrbp1a was separated into six regions based on specific domain features, sequence conserved/polymorphic regions, and the reticulocyte binding like (RBL) domains. In the fragmented gene sequence analysis, PvRBP1a region II (RII) and RIII (head and tail structure homolog, 152–625 aa.) showed extensive polymorphism caused by random point mutations. The haplotype network of these polymorphic regions was classified into three clusters that converged to independent populations. Antigenicity screening was performed using recombinant proteins PvRBP1a-N (157–560 aa.) and PvRBP1a-C (606–962 aa.), which contained head and tail structure region and sequence conserved region, respectively. Sensitivity against PvRBP1a-N (46.7%) was higher than PvRBP1a-C (17.8%). PvRBP1a-N was reported as a reticulocyte binding domain and this study identified a linear epitope with moderate antigenicity, thus an attractive domain for merozoite invasion-blocking vaccine development. However, our study highlights that a global PvRBP1a-based vaccine design needs to overcome several difficulties due to three distinct genotypes and low antigenicity levels.

Genetic diversity of Plasmodium vivax reticulocyte binding protein 2b in global parasite populations

BackgroundPlasmodium vivax reticulocyte binding protein 2b (PvRBP2b) plays a critical role in parasite invasion of reticulocytes by binding the transferrin receptor 1. PvRBP2b is a vaccine candidate based on the negative correlation between antibody titers against PvRBP2b recombinant proteins and parasitemia and risk of vivax malaria. The aim of this study was to analyze the genetic diversity of the PvRBP2b gene in the global P. vivax populations.MethodsNear full-length PvRBP2b nucleotide sequences (190–8349 bp) were obtained from 88 P. vivax isolates collected from the China–Myanmar border (n = 44) and Thailand (n = 44). An additional 224 PvRBP2b sequences were retrieved from genome sequences from parasite populations worldwide. The genetic diversity, neutral selection, haplotype distribution and genetic differentiation of PvRBP2b were examined.ResultsThe genetic diversity of PvRBP2b was distributed unevenly, with peak diversity found in the reticulocyte binding region in the N-terminus. Neutrality analysis suggested that this region is subjected to balancing selection or population bottlenecks. Several amino acid variants were found in all or nearly all P. vivax endemic regions. However, the critical residues responsible for reticulocyte binding were highly conserved. There was substantial population differentiation according to the geographical separation. The distribution of haplotypes in the reticulocyte binding region varied among regions; even the two major haplotypes Hap_6 and Hap_8 were found in only five populations.ConclusionsOur data show considerable genetic variations of PvRBPb in global parasite populations. The geographic divergence may pose a challenge to PvRBP2b-based vaccine development.Graphical

Untangling population structure and genetic diversity of reticulocyte binding protein 2b (PvRBP2b) erythrocytic stage vaccine candidate in worldwide Plasmodium vivax isolates.

BackgroundsPlasmodium vivax is the predominant Plasmodium species distributed extensively in the Americas and Asia-Pacific areas. Encoded protein by Plasmodium vivax Reticulocyte Binding Proteins (PvRBPs) family member are of critical prominence to parasite invasion and have been considered the significant targets in development of malaria vaccine for the blood stage. As high genetic polymorphism of parasites may impede the effectiveness of vaccine development, more research to unraveling genetic polymorphism of pvrbp2b from various geographical regions seems indispensable to map the exact pattern of field isolates.Methodology/Principal findingsThe aim of this study was to determine the sequences of Iranian pvrbp2b (nt: 502–1896) gene and then, to ascertain polymorphism of pvrbp2b gene, recombination, the level of genetic distances, evaluation of natural selection, and the prediction of B-cell epitopes of Iranian and global P. vivax isolates. Pvrbp2b partial gene was amplified and sequenced from 60 Iranian P. vivax isolates. Iranian pvrbp2b sequences as well as 95 published sequences from five countries were used to evaluate the genetic diversity and neutral evolution signature in worldwide scale. A total of 38 SNPs were identified among 60 Iranian pvrbp2b sequences (32 non-synonymous and 6 synonymous mutations), and 32 amino acid substitutions were observed in 29 positions as compared to Sal-1 sequence. Worldwide sequence analysis showed that 44 amino acid changes had occurred in 37 positions of which seven polymorphic sites had trimorphic mutations while the rest was dimorphic. The overall nucleotide diversity for Iranian isolates was 0.00431 ± 0.00091 while the level of nucleotide diversity was ranged from 0.00337 ± 0.00076 (Peru) to 0.00452 ± 0.00092 (Thailand) in global scale.Conclusions/SignificanceOf amino acid substitutions, 12 replacements were located in the B-cell epitopes in which nine polymorphic sites were positioned in N-terminal and three polymorphic sites in predicted B-cell epitopes of C-terminal, signifying both variable and conserved epitopes for vaccine designing. Using the achieved outcome of the current investigation interrogate questions to the selection of conserved regions of pvrbp2b and understanding polymorphism and immune system pressure to pave a way for developing a vaccine based on PvRBP2b candidate antigen.

Comparison of total immunoglobulin G antibody responses to different protein fragments of Plasmodium vivax Reticulocyte binding protein 2b

BackgroundPlasmodium vivax is emerging as the dominant and prevalent species causing malaria in near-elimination settings outside of Africa. Hypnozoites, the dormant liver stage parasite of P. vivax, are undetectable to any currently available diagnostic test, yet are a major reservoir for transmission. Advances have been made to harness the naturally acquired immune response to identify recent exposure to P. vivax blood-stage parasites and, therefore, infer the presence of hypnozoites. This in-development diagnostic is currently able to detect infections within the last 9-months with 80% sensitivity and 80% specificity. Further work is required to optimize protein expression and protein constructs used for antibody detection.MethodsThe antibody response against the top performing predictor of recent infection, P. vivax reticulocyte binding protein 2b (PvRBP2b), was tested against multiple fragments of different sizes and from different expression systems. The IgG induced against the recombinant PvRBP2b fragments in P. vivax infected individuals was measured at the time of infection and in a year-long observational cohort; both conducted in Thailand.ResultsThe antibody responses to some but not all different sized fragments of PvRBP2b protein are highly correlated with each other, significantly higher 1-week post-P. vivax infection, and show potential for use as predictors of recent P. vivax infection.ConclusionsTo achieve P. vivax elimination goals, novel diagnostics are required to aid in detection of hidden parasite reservoirs. PvRBP2b was previously shown to be the top candidate for single-antigen classification of recent P. vivax exposure and here, it is concluded that several alternative recombinant PvRBP2b fragments can achieve equal sensitivity and specificity at predicting recent P. vivax exposure.

The presence of circulating antibody secreting cells and long-lived memory B cell responses to reticulocyte binding protein 1a in Plasmodium vivax patients

BackgroundDevelopment of an effective vaccine against blood-stage malaria requires the induction of long-term immune responses. Plasmodium vivax Reticulocyte Binding Protein 1a (PvRBP1a) is a blood-stage parasite antigen which is associated with invasion of red blood cells and induces antibody responses. Thus, PvRBP1a is considered as a target for design of a blood-stage vaccine against vivax malaria.MethodsBoth cross-sectional and cohort studies were used to explore the development and persistence of long-lived antibody and memory B cell responses to PvRBP1a in individuals who lived in an area of low malaria endemicity. Antibody titers and frequency of memory B cells specific to PvRBP1a were measured during infection and following recovery for up to 12 months.ResultsIgG antibody responses against PvRBP1a were prevalent during acute vivax malaria, predominantly IgG1 subclass responses. High responders to PvRBP1a had persistent antibody responses for at least 12-month post-infection. Further analysis of high responder found a direct relation between antibody titers and frequency of activated and atypical memory B cells. Furthermore, circulating antibody secreting cells and memory B cells specific to PvRBP1a were generated during infection. The PvRBP1a-specific memory B cells were maintained for up to 3-year post-infection, indicating the ability of PvRBP1a to induce long-term humoral immunity.ConclusionThe study revealed an ability of PvRBP1a protein to induce the generation and maintenance of antibody and memory B cell responses. Therefore, PvRBP1a could be considered as a vaccine candidate against the blood-stage of P. vivax.

Naturally induced humoral response against Plasmodium vivax reticulocyte binding protein 2P1

BackgroundPlasmodium vivax is the most prevalent malaria parasite in many countries. A better understanding of human immunity to this parasite can provide new insights for vaccine development. Plasmodium vivax Reticulocyte Binding Proteins (RBPs) are key parasite proteins that interact with human proteins during erythrocyte invasion and are targets of the human immune response. The aim of this study is to characterize the human antibody response to RBP2P1, the most recently described member of the RBP family.MethodsThe levels of total IgG and IgM against RBP2P1 were measured using plasmas from 68 P. vivax malaria patients and 525 villagers in a malarious village of western Thailand. The latter group comprises asymptomatic carriers and healthy uninfected individuals. Subsets of plasma samples were evaluated for anti-RBP2P1 IgG subtypes and complement-fixing activity.ResultsAs age increased, it was found that the level of anti-RBP2P1 IgG increased while the level of IgM decreased. The main anti-RBP2P1 IgG subtypes were IgG1 and IgG3. The IgG3-seropositive rate was higher in asymptomatic carriers than in patients. The higher level of IgG3 was correlated with higher in vitro RBP2P1-mediated complement fixing activity.ConclusionsIn natural infection, the primary IgG response to RBP2P1 was IgG1 and IgG3. The predominance of these cytophilic subtypes and the elevated level of IgG3 correlating with complement fixing activity, suggest a possible role of anti-RBP2P1 antibodies in immunity against P. vivax.

Naturally acquired blocking human monoclonal antibodies to Plasmodiumvivax reticulocyte binding protein 2b

Plasmodium vivax preferentially invades reticulocytes and recognition of these cells is mediated by P. vivax Reticulocyte Binding Protein 2b (PvRBP2b) binding to human Transferrin receptor 1 (TfR1) and Transferrin (Tf). Longitudinal cohort studies in Papua New Guinea, Thailand and Brazil show that PvRBP2b antibodies are correlated with protection against P. vivax infection and disease. Here, we isolate and characterize anti-PvRBP2b human monoclonal antibodies from two individuals in Cambodia with natural P. vivax infection. These antibodies bind with high affinities and map to different regions of PvRBP2b. Several human antibodies block PvRBP2b binding to reticulocytes and inhibit complex formation with human TfR1-Tf. We describe different structural mechanisms for functional inhibition, including either steric hindrance with TfR1-Tf or the reticulocyte membrane. These results show that naturally acquired human antibodies against PvRBP2b can inhibit its function which is important for P. vivax invasion.

Plasmodium vivax Strains Use Alternative Pathways for Invasion.

Plasmodium vivax has 2 invasion ligand/host receptor pathways (P. vivax Duffy-binding protein/Duffy antigen receptor for chemokines [DARC] and P. vivax reticulocyte binding protein 2b/transferrin receptor [TfR1]) that are promising targets for therapeutic intervention. We optimized invasion assays with isogenic cultured reticulocytes. Using a receptor blockade approach with multiple P. vivax isolates, we found that all strains utilized both DARC and TfR1, but with significant variation in receptor usage. This suggests that P. vivax, like Plasmodium falciparum, uses alternative invasion pathways, with implications for pathogenesis and vaccine development.

Plasmodium vivax Reticulocyte Binding Proteins for invasion into reticulocytes.

Plasmodium vivax is responsible for most of the malaria infections outside Africa and is currently the predominant malaria parasite in countries under elimination programs. P. vivax preferentially enters young red cells called reticulocytes. Advances in understanding the molecular and cellular mechanisms of entry are hampered by the inability to grow large numbers of P. vivax parasites in a long‐term in vitro culture. Recent progress in understanding the biology of the P. vivax Reticulocyte Binding Protein (PvRBPs) family of invasion ligands has led to the identification of a new invasion pathway into reticulocytes, an understanding of their structural architecture and PvRBPs as targets of the protective immune response to P. vivax infection. This review summarises current knowledge on the role of reticulocytes in P. vivax infection, the function of the PvRBP family of proteins in generating an immune response in human populations, and the characterization of anti‐PvRBP antibodies in blocking parasite invasion.

Antibodies to Plasmodium vivax reticulocyte binding protein 2b are associated with protection against P. vivax malaria in populations living in low malaria transmission regions of Brazil and Thailand.

BackgroundThe Plasmodium vivax Reticulocyte Binding Protein (PvRBP) family is involved in red blood cell recognition and members of this family are potential targets for antibodies that may block P. vivax invasion. To date, the acquisition of immunity against PvRBPs in low malaria transmission settings and in a broad age group of exposed individuals has not been investigated.Methodology/Principal findingsTotal IgG antibody levels to six members of the PvRBP family (PvRBP1a, PvRBP1b, PvRBP2a, PvRBP2b, a non-binding fragment of PvRBP2c (PvRBP2cNB) and PvRBP2-P2) were measured in samples collected from individuals living in two regions of low P. vivax endemicity in Brazil and Thailand. In both settings, levels of total IgG to PvRBP1a, PvRBP2b, PvRBP2cNB, and PvRBP2P-2 increased significantly with age (rho = 0.17–0.49; P<0.001). IgG responses to PvRBP1a, PvRBP2b and PvRBP2cNB were significantly higher in infected individuals by using Wilcoxon’s signed-rank test (P<0.001). Of the six PvRBPs examined, only antibodies to PvRBP2b were associated with protection against clinical malaria in both settings.Conclusion/SignificanceOur results indicate that PvRBP2b warrants further preclinical development as a blood-stage vaccine candidate against P. vivax. Total IgG responses to PvRBPs were also shown to be promising immunological markers of exposure to P. vivax infection.

Complement Receptor 1 availability on red blood cell surface modulates Plasmodium vivax invasion of human reticulocytes

Plasmodium vivax parasites preferentially invade reticulocyte cells in a multistep process that is still poorly understood. In this study, we used ex vivo invasion assays and population genetic analyses to investigate the involvement of complement receptor 1 (CR1) in P. vivax invasion. First, we observed that P. vivax invasion of reticulocytes was consistently reduced when CR1 surface expression was reduced through enzymatic cleavage, in the presence of naturally low-CR1-expressing cells compared with high-CR1-expressing cells, and with the addition of soluble CR1, a known inhibitor of P. falciparum invasion. Immuno-precipitation experiments with P. vivax Reticulocyte Binding Proteins showed no evidence of complex formation. In addition, analysis of CR1 genetic data for worldwide human populations with different exposure to malaria parasites show significantly higher frequency of CR1 alleles associated with low receptor expression on the surface of RBCs and higher linkage disequilibrium in human populations exposed to P. vivax malaria compared with unexposed populations. These results are consistent with a positive selection of low-CR1-expressing alleles in vivax-endemic areas. Collectively, our findings demonstrate that CR1 availability on the surface of RBCs modulates P. vivax invasion. The identification of new molecular interactions is crucial to guiding the rational development of new therapeutic interventions against vivax 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.

Cryo-EM structure of an essential Plasmodium vivax invasion complex.

Plasmodium vivax is the most widely distributed malaria parasite that infects humans1. P. vivax invades reticulocytes exclusively, and successful entry depends on specific interactions between the P. vivax reticulocyte-binding protein 2b (PvRBP2b) and transferrin receptor 1 (TfR1)2. TfR1-deficient erythroid cells are refractory to invasion by P. vivax, and anti-PvRBP2b monoclonal antibodies inhibit reticulocyte binding and block P. vivax invasion in field isolates2. Here we report a high-resolution cryo-electron microscopy structure of a ternary complex of PvRBP2b bound to human TfR1 and transferrin, at 3.7 Å resolution. Mutational analyses show that PvRBP2b residues involved in complex formation are conserved; this suggests that antigens could be designed that act across P. vivax strains. Functional analyses of TfR1 highlight how P. vivax hijacks TfR1, an essential housekeeping protein, by binding to sites that govern host specificity, without affecting its cellular function of transporting iron. Crystal and solution structures of PvRBP2b in complex with antibody fragments characterize the inhibitory epitopes. Our results establish a structural framework for understanding how P. vivax reticulocyte-binding protein engages its receptor and the molecular mechanism of inhibitory monoclonal antibodies, providing important information for the design of novel vaccine candidates.

Identification and Immunological Characterization of the Ligand Domain of Plasmodium vivax Reticulocyte Binding Protein 1a.

Erythrocyte invasion by malaria parasites is essential for blood-stage development. Consequently, parasite proteins critically involved in erythrocyte invasion, such as the Plasmodium vivax reticulocyte binding proteins (RBPs) that mediate preferential invasion of reticulocytes, are considered potential vaccine targets. Thus, targeting the RBPs could prevent blood-stage infection and disease. The RBPs are large, and little is known about their functional domains and whether individuals naturally exposed to P. vivax acquire binding-inhibitory antibodies to these critical binding regions. This study aims to functionally and immunologically characterize Plasmodium vivax RBP1a. Recombinant proteins of overlapping fragments of RBP1a were used to determine binding specificity to erythrocytes and immunogenicity in laboratory animals. The naturally acquired antibody response to these proteins was evaluated using serum samples from individuals in regions of endemicity. The N-terminal extracellular region, RBP1157-650 (RBP1:F8), was determined to bind both reticulocytes and normocytes, with a preference for immature reticulocytes. Antibodies elicited against rRBP1:F8 blocked binding between RBP1:F8 and erythrocytes. Naturally acquired anti-RBP1 binding-inhibitory antibodies were detected in serum specimens from P. vivax-exposed individuals from Papua New Guinea and Brazil. Recombinant RBP1:F8 binds human erythrocytes, elicits artificially induced functional blocking antibodies, and is a target of naturally acquired binding-inhibitory antibodies.

Naturally Acquired Human Antibodies Against Reticulocyte-Binding Domains of Plasmodium vivax Proteins, PvRBP2c and PvRBP1a, Exhibit Binding-Inhibitory Activity.

Crucial gaps in our understanding of Plasmodium vivax reticulocyte invasion and protective immunity have hampered development of vivax vaccines. P. vivax exclusively invades reticulocytes that is mediated by the P. vivax reticulocyte-binding proteins (PvRBPs) specifically PvRBP2c and PvRBP1a. Vivax infections in Duffy-null individuals have suggested the evolution of alternate invasion pathways that may be mediated by the PvRBPs. Thus, PvRBPs appear as potential targets for efficacious P. vivax neutralization. However, there are limited data validating their vaccine efficacy. In the absence of vivax invasion assays, binding-inhibitory activity of antibodies has been reported to be associated with protection and a measure of vaccine potential. -based analysis was performed of the PvRBP reticulocyte-binding properties and binding-inhibitory activity of specific anti-PvRBP2c/PvRBP1a human antibodies. PvRBP2c and PvRBP1a displayed a distinct reticulocyte-binding specificity, and their specific reticulocyte-binding domains were mapped within their N-terminal regions. Importantly, naturally acquired antibodies against the reticulocyte-binding domains efficaciously blocked reticulocyte binding of native PvRBPs, suggesting that the human immune system produced functional binding-inhibitory antibodies through exposure to vivax malaria. Reticulocyte-binding domains of PvRBP2c/PvRBP1a are targets of naturally acquired binding-inhibitory antibodies, substantiating their promise as candidate antigens against which vaccine-inducible immunity could potentially be boosted through natural infections.

Plasmodium vivax Reticulocyte Binding Proteins Are Key Targets of Naturally Acquired Immunity in Young Papua New Guinean Children.

BackgroundMajor gaps in our understanding of Plasmodium vivax biology and the acquisition of immunity to this parasite hinder vaccine development. P. vivax merozoites exclusively invade reticulocytes, making parasite proteins that mediate reticulocyte binding and/or invasion potential key vaccine or drug targets. While protein interactions that mediate invasion are still poorly understood, the P. vivax Reticulocyte-Binding Protein family (PvRBP) is thought to be involved in P. vivax restricted host-cell selectivity.Methodology/Principal findingsWe assessed the binding specificity of five members of the PvRBP family (PvRBP1a, PvRBP1b, PvRBP2a, PvRBP2b, PvRBP2-P2 and a non-binding fragment of PvRBP2c) to normocytes or reticulocytes. PvRBP2b was identified as the only reticulocyte-specific binder (P<0.001), whereas the others preferentially bound to normocytes (PvRBP1a/b P≤0.034), or showed comparable binding to both (PvRBP2a/2-P2, P = 0.38). Furthermore, we measured levels of total and IgG subclasses 1, 2, 3 and 4 to the six PvRBPs in a cohort of young Papua New Guinean children, and assessed their relationship with prospective risk of P. vivax malaria. Children had substantial, highly correlated (rho = 0.49–0.82, P<0.001) antibody levels to all six PvRBPs, with dominant IgG1 and IgG3 subclasses. Both total IgG (Incidence Rate Ratio [IRR] 0.63–0.73, P = 0.008–0.041) and IgG1 (IRR 0.56–0.69, P = 0.001–0.035) to PvRBP2b and PvRBP1a were strongly associated with reduced risk of vivax-malaria, independently of age and exposure.Conclusion/SignificanceThese results demonstrate a diversity of erythrocyte-binding phenotypes of PvRBPs, indicating binding to both reticulocyte-specific and normocyte-specific ligands. Our findings provide further insights into the naturally acquired immunity to P. vivax and highlight the importance of PvRBP proteins as targets of naturally acquired humoral immunity. In-depth studies of the role of PvRBPs in P. vivax invasion and functional validation of the role of anti-PvRBP antibodies in clinical immunity against P. vivax are now required to confirm the potential of the reticulocyte-binding PvRBP2b and PvRBP1a as vaccine candidate antigens.

Identification of a reticulocyte-specific binding domain of Plasmodium vivax reticulocyte-binding protein 1 that is homologous to the PfRh4 erythrocyte-binding domain.

The Plasmodium vivax reticulocyte-binding protein (RBP) family was identified based on the annotation of adhesive ligands in the P. vivax genome. Reticulocyte-specific interactions with the PvRBPs (PvRBP1 and PvRBP2) were previously reported. Plasmodium falciparum reticulocyte-binding protein homologue 4 (PfRh4, a homologue of PvRBP1) was observed to possess erythrocyte-binding activity via complement receptor 1 on the erythrocyte surface. However, the reticulocyte-binding mechanisms of P. vivax are unclear because of the large molecular mass of PvRBP1 (>326 kDa) and the difficulty associated with in vitro cultivation. In the present study, 34 kDa of PvRBP1a (PlasmoDB ID: PVX_098585) and 32 kDa of PvRBP1b (PVX_098582) were selected from a 30 kDa fragment of PfRh4 for reticulocyte-specific binding activity analysis. Both PvRBP1a and PvRBP1b were found to be localized at the microneme in the mature schizont-stage parasites. Naturally acquired immune responses against PvRBP1a-34 and PvRBP1b-32 were observed lower than PvDBP-RII. The reticulocyte-specific binding activities of PvRBP1a-34 and PvRBP1b-32 were significantly higher than normocyte binding activity and were significantly reduced by chymotrypsin treatment. PvRBP1a and 1b, bind to reticulocytes and that this suggests that these ligands may have an important role in P. vivax merozoite invasion.

Gene Models, Expression Repertoire, and Immune Response of Plasmodium vivax Reticulocyte Binding Proteins.

Members of the Plasmodium vivax reticulocyte binding protein (PvRBP) family are believed to mediate specific invasion of reticulocytes by P. vivax. In this study, we performed molecular characterization of genes encoding members of this protein family. Through cDNA sequencing, we constructed full-length gene models and verified genes that are protein coding and those that are pseudogenes. We also used quantitative PCR to measure their in vivo transcript abundances in clinical P. vivax isolates. Like genes encoding related invasion ligands of P. falciparum, Pvrbp expression levels vary broadly across different parasite isolates. Through antibody measurements, we found that host immune pressure may be the driving force behind the distinctly high diversity of one of the family members, PvRBP2c. Mild yet significant negative correlation was found between parasitemia and the PvRBP2b antibody level, suggesting that antibodies to the protein may interfere with invasion.

Identification of Immunodominant B-cell Epitope Regions of Reticulocyte Binding Proteins in Plasmodium vivax by Protein Microarray Based Immunoscreening.

Plasmodium falciparum can invade all stages of red blood cells, while Plasmodium vivax can invade only reticulocytes. Although many P. vivax proteins have been discovered, their functions are largely unknown. Among them, P. vivax reticulocyte binding proteins (PvRBP1 and PvRBP2) recognize and bind to reticulocytes. Both proteins possess a C-terminal hydrophobic transmembrane domain, which drives adhesion to reticulocytes. PvRBP1 and PvRBP2 are large (> 326 kDa), which hinders identification of the functional domains. In this study, the complete genome information of the P. vivax RBP family was thoroughly analyzed using a prediction server with bioinformatics data to predict B-cell epitope domains. Eleven pvrbp family genes that included 2 pseudogenes and 9 full or partial length genes were selected and used to express recombinant proteins in a wheat germ cell-free system. The expressed proteins were used to evaluate the humoral immune response with vivax malaria patients and healthy individual serum samples by protein microarray. The recombinant fragments of 9 PvRBP proteins were successfully expressed; the soluble proteins ranged in molecular weight from 16 to 34 kDa. Evaluation of the humoral immune response to each recombinant PvRBP protein indicated a high antigenicity, with 38-88% sensitivity and 100% specificity. Of them, N-terminal parts of PvRBP2c (PVX_090325-1) and PvRBP2 like partial A (PVX_090330-1) elicited high antigenicity. In addition, the PvRBP2-like homologue B (PVX_116930) fragment was newly identified as high antigenicity and may be exploited as a potential antigenic candidate among the PvRBP family. The functional activity of the PvRBP family on merozoite invasion remains unknown.

Plasmodium falciparum Reticulocyte Binding-Like Homologue Protein 2 (PfRH2) Is a Key Adhesive Molecule Involved in Erythrocyte Invasion

Erythrocyte invasion by Plasmodium merozoites is a complex, multistep process that is mediated by a number of parasite ligand-erythrocyte receptor interactions. One such family of parasite ligands includes the P. falciparum reticulocyte binding homologue (PfRH) proteins that are homologous with the P. vivax reticulocyte binding proteins and have been shown to play a role in erythrocyte invasion. There are five functional PfRH proteins of which only PfRH2a/2b have not yet been demonstrated to bind erythrocytes. In this study, we demonstrated that native PfRH2a/2b is processed near the N-terminus yielding fragments of 220 kDa and 80 kDa that exhibit differential erythrocyte binding specificities. The erythrocyte binding specificity of the 220 kDa processed fragment of native PfRH2a/2b was sialic acid-independent, trypsin resistant and chymotrypsin sensitive. This specific binding phenotype is consistent with previous studies that disrupted the PfRH2a/2b genes and demonstrated that PfRH2b is involved in a sialic acid independent, trypsin resistant, chymotrypsin sensitive invasion pathway. Interestingly, we found that the smaller 80 kDa PfRH2a/2b fragment is processed from the larger 220 kDa fragment and binds erythrocytes in a sialic acid dependent, trypsin resistant and chymotrypsin sensitive manner. Thus, the two processed fragments of PfRH2a/2b differed with respect to their dependence on sialic acids for erythrocyte binding. Further, we mapped the erythrocyte binding domain of PfRH2a/2b to a conserved 40 kDa N-terminal region (rPfRH240) in the ectodomain that is common to both PfRH2a and PfRH2b. We demonstrated that recombinant rPfRH240 bound human erythrocytes with the same specificity as the native 220 kDa processed protein. Moreover, antibodies generated against rPfRH240 blocked erythrocyte invasion by P. falciparum through a sialic acid independent pathway. PfRH2a/2b thus plays a key role in erythrocyte invasion and its conserved receptor-binding domain deserves attention as a promising candidate for inclusion in a blood-stage malaria vaccine.