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Abstract

Plasmodium vivax merozoite preferentially invades reticulocytes probably using PvRBP-1 as ligand. One hundred and ninety-five, 15-mer peptides has been synthesised from PvRBP-1 sequence; tested in reticulocyte- or erythrocyte-binding assays. Twenty-five peptides (K(d)=76-380 nM) specifically defined four reticulocyte-binding regions. It has been reported that a highly conserved Region-I recombinant fragment binds specifically to reticulocytes. HABP-critical residues for reticulocyte-binding were highly conserved in 20 Colombian P. vivax clinical isolates, suggesting an important biological function. There were six overlapping reticulocyte-binding sites for these peptides according to enzyme sensitivity and mutual competition-binding assays; located on 26- and 41-kDa reticulocyte membrane surface proteins.

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... However, the mechanisms underlying the molecular interactions between parasite ligands and host-cell receptors during reticulocyte invasion remain unclear. Comparion of P. falciparum reticulocyte binding-like proteins (PfRhs) [9][10][11][12][13][14][15][16][17][18][19], P. vivax reticulocyte binding-like proteins (PvRBPs) have not been thoroughly investigated [20][21][22][23][24]. ...
... Thus, fragments covering the entire sequence were constructed and contained a region at the N-terminals of both proteins that exhibited reticulocyte-binding activity [49]. Furthermore, analyses of overlapping 15-mer PvRBP1 synthetic peptides indicated the existence of four reticulocyte-binding regions (RBR1-I to -IV), which showed high-affinity binding to reticulocytes [22] (Fig. 3). RBR1-III showed the most reticulocyte-binding sequences [22]. ...
... Furthermore, analyses of overlapping 15-mer PvRBP1 synthetic peptides indicated the existence of four reticulocyte-binding regions (RBR1-I to -IV), which showed high-affinity binding to reticulocytes [22] (Fig. 3). RBR1-III showed the most reticulocyte-binding sequences [22]. Nevertheless, synthetic peptides have limitations compared to native peptides, such as a lack of post-translational modifications and conformational motifs, which hamper further functional study. ...
... Unfortunately, basic P. vivax research has been delayed mainly due to the parasite's preference for invading reticulocytes which are difficult to obtain in the high percentages needed for propagating P. vivax in vitro [12,13]. However, it has been possible to characterise several molecules forming part of the parasite's selective human reticulocyte invasion route, such as reticulocyte binding proteins (RBPs) [14,15], merozoite surface protein 1 (MSP-1) [16], some proteins from the tryptophanrich antigen (TRAg) family [17] and the recently described rhoptry neck protein 5 (RON5) [18]. Some of these contain specific binding regions that have been identified using several strategies, such as mapping using peptides labelled with radioactive iodine, ELISA, flow cytometry or rosetting assays. ...
... ± 3.3) can be the explanation of why PvGAMA fragment binding to 100% of the CD71+ reticulocytes was not found (Fig. 4a). It has been observed that several P. vivax proteins, such as DBP [64], MSP-1 [16], RBP1 [14], the erythrocyte binding protein (EBP) [42], RBP1a, RBP1b [65] and RBP2 [15], have preferential reticulocyte binding activity, being the RBPs particularly important in parasite cell selection. Taking the results obtained here into account, it can be suggested that P. vivax target cell selection is not only governed by the RBPs but other ligands are also taking place in this process, such as DBP, MSP-1, EBP and now PvGAMA. ...
Article
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Background Adhesin proteins are used by Plasmodium parasites to bind and invade target cells. Hence, characterising molecules that participate in reticulocyte interaction is key to understanding the molecular basis of Plasmodium vivax invasion. This study focused on predicting functionally restricted regions of the P. vivax GPI-anchored micronemal antigen (PvGAMA) and characterising their reticulocyte binding activity. Results The pvgama gene was initially found in P. vivax VCG-I strain schizonts. According to the genetic diversity analysis, PvGAMA displayed a size polymorphism very common for antigenic P. vivax proteins. Two regions along the antigen sequence were highly conserved among species, having a negative natural selection signal. Interestingly, these regions revealed a functional role regarding preferential target cell adhesion. Conclusions To our knowledge, this study describes PvGAMA reticulocyte binding properties for the first time. Conserved functional regions were predicted according to natural selection analysis and their binding ability was confirmed. These findings support the notion that PvGAMA may have an important role in P. vivax merozoite adhesion to its target cells. Electronic supplementary material The online version of this article (doi:10.1186/s13071-017-2183-8) contains supplementary material, which is available to authorized users.
... High affinity reticulocyte-binding peptides (HARBPs) have been identified, 5 in a fragment from the region I of PvRBP1 NT extreme (222) and 24 throughout the whole protein (223). The highly-conserved region III (between amino acids 1,941-2,229) had the greatest amount of HARBPs (223) and, when used as immunogen, it induced high antibody titers in Aotus nancymaae monkeys, able to recognize the full PvRBP1 in parasite lysate. ...
... High affinity reticulocyte-binding peptides (HARBPs) have been identified, 5 in a fragment from the region I of PvRBP1 NT extreme (222) and 24 throughout the whole protein (223). The highly-conserved region III (between amino acids 1,941-2,229) had the greatest amount of HARBPs (223) and, when used as immunogen, it induced high antibody titers in Aotus nancymaae monkeys, able to recognize the full PvRBP1 in parasite lysate. T-lymphocytes became activated following the second and third doses, but no protection was obtained after experimental challenge (224). ...
Article
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Malaria caused by Plasmodium vivax continues being one of the most important infectious diseases around the world; P. vivax is the second most prevalent species and has the greatest geographic distribution. Developing an effective antimalarial vaccine is considered a relevant control strategy in the search for means of preventing the disease. Studying parasite-expressed proteins, which are essential in host cell invasion, has led to identifying the regions recognized by individuals who are naturally exposed to infection. Furthermore, immunogenicity studies have revealed that such regions can trigger a robust immune response that can inhibit sporozoite (hepatic stage) or merozoite (erythrocyte stage) invasion of a host cell and induce protection. This review provides a synthesis of the most important studies to date concerning the antigenicity and immunogenicity of both synthetic peptide and recombinant protein candidates for a vaccine against malaria produced by P. vivax.
... RBP-1 is a member of a cell-binding protein family identified in P. vivax; in the Sal-I strain, this family has 10 rbp genes including three partial genes (one rbp- [5]. Mapping the whole of P. vivax Belem strain rbp-1 (encoded by the rbp-1a gene) has led to the definition of four reticulocyte-binding regions (RI-RIV) housing several highly conserved high-binding peptides (Figure 3) [38]. A further study has shown that RBP-1 region I, recombinantly expressed in Spodoptera frugiperda (Sf9) insect cell line, intervenes in merozoites' specific binding to reticulocytes [39]. ...
... Based on the strength of FIDIC's studies involving the molecular characterization of 50 proteins implicated in P. falciparum invasion, advances have been made in screening three P. vivax molecules. These antigens' regions for specific binding to target cells have been determined by using enriched reticulocytes from samples from a patient suffering from β-thalassemia and synthetic peptides; DBP [28], RBP-1 [38] and MSP-1 [66] have thus been fully mapped (Figure 3). Such advances have provided valuable knowledge for developing novel strategies and methodologies providing in-depth knowledge about the importance of candidates and their inclusion in an epitope-based vaccine. ...
Article
Malaria caused by Plasmodium vivax continues being a public health problem in tropical and subtropical areas throughout the whole world. In spite of this species' epidemiological importance, its biological complexity has hampered advances being made in the field of vaccine development. Few antigens have been described and analyzed to date in preclinical and clinical studies, thereby highlighting the great challenge facing groups currently working on this parasite species. This review summarizes the most representative work done during the last few years and discusses the approaches adopted in making progress towards an anti-Plasmodium vivax vaccine.
... PvRBP1 also has two RGD motifs which could have a reticulocyte binding function. Urquiza et al. described four regions within the PvRBP1 sequence containing 25 high reticulocyte binding peptides [16]; the so-called region III, comprising amino acids (aa) 1998-2348, was the most conserved. It also contained nine high reticulocyte binding peptides which displayed greater binding activity than peptides from the other regions. ...
... It was decided to work with a recombinant segment of the PvRBP1 from the so-called region III due to this protein's great importance in reticulocyte invasion and the urgent need to find new antigens which might be included in an effective vaccine against P. vivax. Region III has been shown to have high reticulocyte binding ability, containing nine high binding activity peptides [16]. ...
Article
Plasmodium vivax merozoites have been found to specifically invade immature red blood cells (reticulocytes) and this preference has been associated with two proteins named reticulocyte binding protein-1 and protein-2 (PvRBP1 and PvRBP2). Previous reticulocyte binding assays using 15-mer synthetic peptides spanning the entire PvRBP1 sequence have shown that 25 out of the 195 peptides synthesised (grouped into 4 different regions) displayed high affinity binding to reticulocytes. The PvRBP1 region III (amino acids 1998-2348), encompassing 9 of the previously described high-affinity reticulocyte binding peptides, was expressed as a recombinant protein in the present study. This protein has been shown to be antigenic in humans and it has also been able to induce good humoral and cellular immune responses in Aotus nancymaae monkeys. Despite its high immunogenicity, no protective efficacy was observed in the immunised animals.
... IgG prevalence against the NT region (including the most polymorphic region) of the PvRBP1a and b was intermediate in a population from Thailand [117], but IgG prevalence against the PvRBP1a-34 and PvRBP1b-32 proteins was significantly higher in P. vivax patients than healthy individuals in the Republic of Korea [83]. Moreover, the highly conserved region III (between amino acids 1941-2229), with the greatest amount of high-affinity reticulocytebinding peptides and high binding affinity, was shown to induce high antibody titers in Aotus monkeys and be able to recognize the full PvRBP1 in parasite lysate [157]. The expression of PvRBPs in the African P. vivax and the antibody response against PvRBP in Duffy-negatives will provide important implications to the usefulness of a future vaccine in vivax malaria control in Africa. ...
Article
Full-text available
Plasmodium vivax malaria is a neglected tropical disease, despite being more geographically widespread than any other form of malaria. The documentation of P. vivax infections in different parts of Africa where Duffy-negative individuals are predominant suggested that there are alternative pathways for P. vivax to invade human erythrocytes. Duffy-negative individuals may be just as fit as Duffy-positive individuals and are no longer resistant to P.vivax malaria. In this review, we describe the complexity of P. vivax malaria, characterize pathogenesis and candidate invasion genes of P. vivax, and host immune responses to P. vivax infections. We provide a comprehensive review on parasite ligands in several Plasmodium species that further justify candidate genes in P. vivax. We also summarize previous genomic and transcriptomic studies related to the identification of ligand and receptor proteins in P. vivax erythrocyte invasion. Finally, we identify topics that remain unclear and propose future studies that will greatly contribute to our knowledge of P. vivax.
... IgG prevalence against the NT region (including the most polymorphic region) of the PvRBP1a and b was intermediate in a population from Thailand [114], but IgG prevalence against the PvRBP1a-34 and PvRBP1b-32 proteins was significantly higher in P. vivax patients than healthy individuals in the Republic of Korea [80]. Also, the highly-conserved region III (between amino acids 1,941-2,229) with the greatest amount of high-affinity reticulocytebinding peptides and high binding affinity was shown to induce high antibody titers in Aotus monkeys and able to recognize the full PvRBP1 in parasite lysate [154]. repeat and C-terminal regions [155]. ...
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Plasmodium vivax malaria is a neglected tropical disease, despite being more geographically widespread than any other form of malaria. The documentation of P. vivax infections in different parts of Africa where Duffy-negative individuals are predominant suggested that there are alternative pathways for P. vivax to invade human erythrocytes. Duffy-negative individuals may be just as fit as Duffy-positive individuals and are no longer resistant to P. vivax malaria. In this review, we describe the complexity of P. vivax malaria, characterize pathogenesis and candidate invasion genes of P. vivax, and host immune responses to P. vivax infections. We provide a comprehensive review on parasite ligands in several Plasmodium species that further justify candidate genes in P. vivax. We also summarize previous genomic and transcriptomic studies related to the identification of ligand and receptor proteins in P. vivax erythrocyte invasion. Finally, we identify topics that remain unclear and propose future studies that will greatly contribute to our knowledge of P. vivax.
... Mrz proteins' subcellular localization is indicated. An asterisk indicates those molecules with unknown subcellular localization GPI-anchored micronemal antigen (GAMA) [44], reticulocyte binding surface antigen (RBSA) [45], the Duffy binding protein (DBP) (DARC as receptor) [46,47], reticulocyte binding protein 1 (RBP-1) [48], merozoite surface protein 1 (MSP-1) (possible receptor, band 3) [49], apical membrane antigen 1 (AMA-1) (chymotrypsin-and neuraminidase-sensitive receptor, GPB?) [50] and rhoptry neck proteins 2 and 4 (RON2 and RON4) [51]. ...
Article
Full-text available
Abstract Understanding the life cycle of Plasmodium vivax is fundamental for developing strategies aimed at controlling and eliminating this parasitic species. Although advances in omic sciences and high-throughput techniques in recent years have enabled the identification and characterization of proteins which might be participating in P. vivax invasion of target cells, exclusive parasite tropism for invading reticulocytes has become the main obstacle in maintaining a continuous culture for this species. Such advance that would help in defining each parasite protein’s function in the complex process of P. vivax invasion, in addition to evaluating new therapeutic agents, is still a dream. Advances related to maintenance, culture medium supplements and the use of different sources of reticulocytes and parasites (strains and isolates) have been made regarding the development of an in vitro culture for P. vivax; however, only some cultures having few replication cycles have been obtained to date, meaning that this parasite’s maintenance goes beyond the technical components involved. Although it is still not yet clear which molecular mechanisms P. vivax prefers for invading young CD71+ reticulocytes [early maturation stages (I–II–III)], changes related to membrane proteins remodelling of such cells could form part of the explanation. The most relevant aspects regarding P. vivax in vitro culture and host cell characteristics have been analysed in this review to explain possible reasons why the species’ continuous in vitro culture is so difficult to standardize. Some alternatives for P. vivax in vitro culture have also been described.
... To date, PvMSP-1 19 , PvRBP-1a 92 and PvDBP 72 HABPs have been identified which bind specifically to reticulocytes; this, together with characterizing PvAMA-1 regions, has increased current knowledge about P. vivax Mrz specific binding properties used for invading reticulocytes. Future work should address evaluating naturally-acquired antibodies capability for blocking PvAMA-1 binding to reticulocytes and correlate the results with some degree of protection. ...
Article
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The malarial parasite's invasion is complex, active and coordinated, involving many low and high affinity interactions with receptors on target cell membrane. Proteomics analysis has described around 40 proteins in P. vivax which could be involved in reticulocyte invasion; few have been studied with the aim of elucidating how many of them establish specific interactions with their respective host cells. Given the importance of knowing which of the parasite's protein regions are functionally important for invasion, minimum regions mediating specific interaction between Plasmodium vivax apical membrane antigen 1 (PvAMA-1) and its host cell were here elucidated. The region covering PvAMA-1 domains I and II (PvAMA-DI-II) specifically bound to the CD71⁺ red blood cell subpopulation. A 20 residue-long region (⁸¹EVENAKYRIPAGRCPVFGKG¹⁰⁰) located in domain I was capable of inhibiting PvAMA-DI-II recombinant protein binding to young reticulocytes (CD71⁺CD45⁻) and rosette formation. This conserved peptide specifically interacted with high affinity with reticulocytes (CD71⁺) through a neuraminidase-and chymotrypsin-treatment sensitive receptor. Such results showed that, despite AMA-1 having universal functions during late Plasmodium invasion stages, PvAMA-1 had reticulocyte-preferring binding regions, suggesting that P. vivax target cell selection is not just restricted to initial interactions but maintained throughout the erythrocyte invasion cycle, having important implications for designing a specific anti-P. vivax vaccine.
... The Aotus species has led to an enormous advance regarding preclinical studies highlighting the immunological and protective role of various molecules or parts of them from the P. falciparum FVO strain; taking into account that the complex machinery involved in erythrocyte invasion used by this parasite for infecting cells is partly known today, the Aotus model has been essential for describing the fundamental basis when identifying vaccine components against this parasite species [11,12]. On the other hand, these primates develop very reproducible infection following experimental infection with the P. vivax VCG-1 (Vivax Colombia Guaviare-1) strain, having high levels of parasitaemia (N 5%) after 22 passages [13]; this has been of great importance for advancing molecular (MSP-7, Pv38, RAP-1 and RBP-1 between others) [14][15][16][17] and immunological (MSP-1 20 and MSP-1 14 from MSP-1 33 fragment) [18,19] characterisation studies of some molecules from the P. vivax species and evaluating their usefulness in developing an effective vaccine. These findings highlight the fact that using A. nancymaae in combination with the P. falciparum FVO or P. vivax VCG-1 strains is valuable for screening suitable vaccine candidates for later testing in humans. ...
... The lack of a continuous source of reticulocyte-enriched samples regarding P. vivax has hampered the identification of regions where P. vivax antigens bind to their target cells, using the methodology adopted concerning P. falciparum. Binding regions have been identified in three P. vivax proteins to date: PvMSP1, PvDBP and PvRBP-1 [58][59][60]. Two qualitative methodologies have been adopted to make further advances in identifying these regions; they require fewer target cells for evaluating whether the PvRON5 carboxyl terminal region interacts with umbilical cord samples and reticulocyte-enriched samples. Figure 4A shows that rPvRON5 bound to umbilical cord RBC and reticulocyte-enriched samples. ...
Article
Different proteins derived from the membrane or the apical organelles become involved in malarial parasite invasion of host cells. Among these, the rhoptry neck proteins (RONs) interact with a protein component of the micronemes to enable the formation of a strong bond which is crucial for the parasite's successful invasion. The present study was aimed at identifying and characterizing the RON5 protein in Plasmodium vivax and evaluating its ability to bind to reticulocytes. Taking the Plasmodium falciparum and Plasmodium knowlesi RON5 amino acid sequences as template, an in-silico search was made in the P. vivax genome for identifying the orthologous gene. Different molecular tools were used for experimentally ascertaining pvron5 gene presence and transcription in P. vivax VCG-1 strain schizonts. Polyclonal antibodies against PvRON5 peptides were used for evaluating protein expression (by Western blot) and sub-cellular localization (by immunofluorescence). A 33 kDa PvRON5 fragment was expressed in Escherichia coli and used for evaluating the reactivity of sera from patients infected by P. vivax. Two assays were made for determining the RON5 recombinant fragment's ability to bind to reticulocyte-enriched human umbilical cord samples. The pvron5 gene (3,477 bp) was transcribed in VCG-1 strain schizonts and encoded a ~133 kDa protein which was expressed in the rhoptry neck of VCG-1 strain late schizonts, together with PvRON2 and PvRON4. Polyclonal sera against PvRON5 peptides specifically detected ~85 and ~30 kDa fragments in parasite lysate, thereby suggesting proteolytic processing in this protein. Comparative analysis of VCG-1 strain PvRON5 with other P. vivax strains having different geographic localizations suggested its low polymorphism regarding other malarial antigens. A recombinant fragment of the PvRON5 protein (rPvRON5) was recognized by sera from P. vivax-infected patients and bound to red blood cells, having a marked preference for human reticulocytes. The pvron5 gene is transcribed in the VCG-1 strain, the encoded protein is expressed at the parasite's apical pole and might be participating in merozoite invasion of host cells, taking into account its marked binding preference for human reticulocytes.
... There has been little reported regarding the structure and function of the reticulocyte binding-like proteins (RBPs) from P. vivax, PvRBP1 and PvRBP2 (Galinski et al., 1992) or the Plasmodium yoelii homolog Py235 (Holder et al., 1991). PvRBP receptors have yet to be identified, in part because it is not possible to culture blood stages of P. vivax; however, PvRBP1 peptides that bind to reticulocytes have been identified (Cantor et al., 2001;Urquiza et al., 2002). A short review by Li and Han (2012) describes the RBPs in further detail. ...
Article
Full-text available
The apicomplexan family of pathogens, which includes Plasmodium spp. and Toxoplasma gondii, are primarily obligate intracellular parasites and invade multiple cell types. These parasites express extracellular membrane protein receptors, adhesins, to form specific pathogen-host cell interaction complexes. Various adhesins are used to invade a variety of cell types. The receptors are linked to an actomyosin motor, which is part of a complex comprised of many proteins known as the invasion machinery or glideosome. To date, reviews on invasion have focused primarily on the molecular pathways and signals of invasion, with little or no structural information presented. Over 75 structures of parasite receptors and glideosome proteins have been deposited with the Protein Data Bank. These structures include adhesins, motor proteins, bridging proteins, inner membrane complex and cytoskeletal proteins, as well as co-crystal structures with peptides and antibodies. These structures provide information regarding key interactions necessary for target receptor engagement, machinery complex formation, how force is transmitted, and the basis of inhibitory antibodies. Additionally, these structures can provide starting points for the development of antibodies and inhibitory molecules targeting protein-protein interactions, with the aim to inhibit invasion. This review provides an overview of the parasite adhesin protein families, the glideosome components, glideosome architecture, and discuss recent work regarding alternative models. Copyright © 2015 Elsevier Inc. All rights reserved.
... For instance, it has a preference for invading reticulocytes, a small percentage of which are found in peripheral blood [5], making it difficult to standardise an in vitro continuous culture for obtaining large amounts of parasite [5]. Regarding vaccine design, the molecules involved in invasion are highly polymorphic, i.e. the Duffy binding protein (DBP) [6], apical merozoite antigen 1 (AMA-1) [7], reticulocyte binding proteins (RBPs) [8,9] and merozoite surface protein 1 (MSP-1) [10]. The picture is further complicated as latent liver forms (hypnozoites) generate new parasites which are genetically different from those found during the primary infection [11,12]. ...
Article
Biological significance: P. vivax malaria continues being a public health problem around world. Although considerable progress has been made in understanding genome- and transcriptome-related P. vivax biology, there are few proteome studies, currently representing only 8.5% of the predicted in silico proteome reported in public databases. A high-throughput proteomic assay was used for discovering new P. vivax intra-reticulocyte asexual stage molecules taken from parasites maintained in vivo in a primate model. The methodology avoided the main problem related to standardising an in vitro culture system to obtain enough samples for protein identification and annotation. This study provides a source of potential information contributing towards a basic understanding of P. vivax biology related to parasite proteins which are of significant importance for the malaria research community.
... Our group reported using blood from a patient suffering from β-thalassaemia in 2002, demonstrating Review that such blood contained greater than 85% reticulocytes Rodriguez et al., 2002;Urquiza et al., 2002). Samples obtained by venipuncture were washed and passed through a CF11 cellulose column to remove leucocytes and then used for identifying peptide sequences from P. vivax proteins binding with high affinity to target cells. ...
Article
Reticulocytes represent the main invasion target for Plasmodium vivax, the second most prevalent parasite species around the world causing malaria in humans. In spite of these cells importance in research into malaria, biological knowledge related to the nature of the host has been limited, given the technical difficulties present in working with them in the laboratory. Poor reticulocyte recovery from total blood, by different techniques, has hampered continuous in vitro P. vivax cultures being developed, thereby delaying basic investigation in this parasite species. Intense research during the last few years has led to advances being made in developing methodologies orientated towards obtaining enriched reticulocytes from differing sources, thereby providing invaluable information for developing new strategies aimed at preventing infection caused by malaria. This review describes the most recent studies related to obtaining reticulocytes and discusses approaches which could contribute towards knowledge regarding molecular interactions between target cell proteins and their main infective agent, P. vivax.
... The pvrbp2 gene was found to be much more diverse than pvrbp1 in an initial survey of parasite isolates originating from Brazil and Thailand (Rayner et al., 2005). Polymorphisms within pvrbp1 were most abundant in the N-terminal region of this protein, within the location of a predicted RBC-binding domain (Rosas-Acosta, 1998;Urquiza et al., 2002); a similar binding region has been identified in P. falciparum (Gao et al., 2008;Gaur et al., 2007;Rodriguez et al., 2008;Triglia et al., 2011) and P. knowlesi (Semenya et al., 2012) RBL homologues. ...
Article
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Population studies show that individuals acquire immunity to Plasmodium vivax more quickly than Plasmodium falciparum irrespective of overall transmission intensity, resulting in the peak burden of P. vivax malaria in younger age groups. Similarly, actively induced P. vivax infections in malaria therapy patients resulted in faster and generally more strain-transcending acquisition of immunity than P. falciparum infections. The mechanisms behind the more rapid acquisition of immunity to P. vivax are poorly understood. Natural acquired immune responses to P. vivax target both pre-erythrocytic and blood-stage antigens and include humoral and cellular components. To date, only a few studies have investigated the association of these immune responses with protection, with most studies focussing on a few merozoite antigens (such as the Pv Duffy binding protein (PvDBP), the Pv reticulocyte binding proteins (PvRBPs), or the Pv merozoite surface proteins (PvMSP1, 3 & 9)) or the circumsporozoite protein (PvCSP). Naturally acquired transmission-blocking (TB) immunity (TBI) was also found in several populations. Although limited, these data support the premise that developing a multi-stage P. vivax vaccine may be feasible and is worth pursuing.
... 8 kb–9 kb). Thus, overlapping peptides were used to define the binding domains of Pvrbp1 [16], [17], [18]. The other investigation was confined to an assessment of Pvrbp1 and Pvrbp2 diversity in four isolates from different geographical regions [19], which revealed a remarkably high diversity in Pvrbp2 as compared to Pvrbp1 and its homolog in P. falciparum and provided an indication as to the sub-domains it might be worth to focus on in future studies. ...
Article
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Plasmodium vivax merozoites specifically invade reticulocytes. Until recently, two reticulocyte-binding proteins (Pvrbp1 and Pvrbp2) expressed at the apical pole of the P. vivax merozoite were considered to be involved in reticulocyte recognition. The genome sequence recently obtained for the Salvador I (Sal-I) strain of P. vivax revealed additional genes in this family, and in particular Pvrbp2a, Pvrbp2b (Pvrbp2 has been renamed as Pvrbp2c) and two pseudogenes Pvrbp2d and Pvrbp3. It had been previously found that Pvrbp2c is substantially more polymorphic than Pvrbp1. The primary goal of this study was to ascertain the level of polymorphism of these new genes. The sequence of the Pvrbp2a, Pvrbp2b, Pvrbp2d and Pvrbp3 genes were obtained by amplification/cloning using DNA purified from four isolates collected from patients that acquired the infection in the four cardinal regions of Thailand (west, north, south and east). An additional seven isolates from western Thailand were analyzed for gene copy number variation. There were significant polymorphisms exhibited by these genes (compared to the reference Sal-I strain) with the ratio of mutations leading to a non-synonymous or synonymous amino acid change close to 3∶1 for Pvrbp2a and Pvrbp2b. Although the degree of polymorphism exhibited by these two genes was higher than that of Pvrbp1, it did not reach the exceptional diversity noted for Pvrbp2c. It was interesting to note that variations in the copy number of Pvrbp2a and Pvrbp2b occurred in some isolates. The evolution of different members of the Pvrbp2 family and their relatively high degree of polymorphism suggests that the proteins encoded by these genes are important for parasite survival and are under immune selection. Our data also shows that there are highly conserved regions in rbp2a and rbp2b, which might provide suitable targets for future vaccine development against the blood stage of P. vivax.
... The 46 proteins identified in the target data set (45 positive and 1 negative) were compared against those reported in the literature and analyzed in terms of several structural features such as the presence of putative classical and non-classical signal sequences, the number of transmembrane helices, the presence of GPIanchor sites, as well as the presence of protein domains relevant for invasion of erythrocytes and able to mediate cell adhesion ( Table 3). The final categorization included 13 proteins which had already been classified as vaccine candidates according to previous studies; 9 of these have been tested for their antigenicity [21][22][23][24][25], two have been assayed for their protection-inducing ability [5,21,26,27] and strong reticulocyte-binding ability has been described for the remaining two [28,29]. Experimental evidence of protein expression has been published for 7 additional ORFs; however, no immunological or functional assays testing their potential role as vaccine candidates have been reported yet [5,[30][31][32]. ...
Article
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This study describes a bioinformatics approach designed to identify Plasmodium vivax proteins potentially involved in reticulocyte invasion. Specifically, different protein training sets were built and tuned based on different biological parameters, such as experimental evidence of secretion and/or involvement in invasion-related processes. A profile-based sequence method supported by hidden Markov models (HMMs) was then used to build classifiers to search for biologically-related proteins. The transcriptional profile of the P. vivax intra-erythrocyte developmental cycle was then screened using these classifiers. A bioinformatics methodology for identifying potentially secreted P. vivax proteins was designed using sequence redundancy reduction and probabilistic profiles. This methodology led to identifying a set of 45 proteins that are potentially secreted during the P. vivax intra-erythrocyte development cycle and could be involved in cell invasion. Thirteen of the 45 proteins have already been described as vaccine candidates; there is experimental evidence of protein expression for 7 of the 32 remaining ones, while no previous studies of expression, function or immunology have been carried out for the additional 25. The results support the idea that probabilistic techniques like profile HMMs improve similarity searches. Also, different adjustments such as sequence redundancy reduction using Pisces or Cd-Hit allowed data clustering based on rational reproducible measurements. This kind of approach for selecting proteins with specific functions is highly important for supporting large-scale analyses that could aid in the identification of genes encoding potential new target antigens for vaccine development and drug design. The present study has led to targeting 32 proteins for further testing regarding their ability to induce protective immune responses against P. vivax malaria.
... The methodology used here for identifying binding fragments from the selected proteins has been previously applied in P. falciparum studies [4,28,30,31,44,55,68,69], Plasmodium vivax [48,67] and M. tuberculosis [12,18,26,49,50,53,58,70]. In the case of P. falciparum, identified sequences have been the base to the ratio-nal design of subunit-based, multiantigenic, multistage chemically synthesized vaccines, and a universal methodology has been proposed, that might be applied to prevent different infectious diseases [13,51,52]. For M. tuberculosis proteins, and specifically for Rv0180c, identified HABPs capable of inhibiting mycobacterial entry to host cells could be included in immunological studies using animal models, to assess their potential as components of a subunit-based anti-tuberculous vaccine. ...
Article
The identification and characterization of hypothetical membrane proteins from Mycobacterium tuberculosis have led to a better understanding of the mechanisms used by this pathogen to invade and survive inside host cells. This study assessed the presence, transcription, localization and possible biological activity of the conserved hypothetical protein Rv0180c from M. tuberculosis. Bioinformatics analyses indicated that Rv0180c contains a signal peptide, six possible transmembrane helices and a Plasmodium Export Element (PEXEL)-like motif. PCR analyses showed the presence of the Rv0180c gene in strains from the M. tuberculosis complex; but transcription was not detected in Mycobacterium microti. Sera against synthetic peptides of Rv0180c recognized two protein bands in M. tuberculosis H37Rv sonicate: a ∼48-kDa band close to the predicted molecular mass of Rv0180c (47.6 kDa), and a 63-kDa band probably caused by protein modifications. Moreover, the same sera located the protein on the surface of M. tuberculosis H37Rv bacilli by immunoelectron microscopy. Twenty-three synthetic peptides spanning the entire length of Rv0180c were tested for their ability to bind to U937 and A549 cells, finding nine high-activity binding peptides (HABPs) specific for both cell types, two HABPs specific for A549 cells (namely 31032 and 31044) and two HABPs specific for U937 cells (namely 31025 and 31041). HABPs inhibited invasion of M. tuberculosis H37Rv into A549 or U937 cells by significant percentages and facilitated internalization of latex beads in A549 cells. The Rv0180c HABPs herein reported could be preliminary candidates to be assessed as components of a multiepitope, chemically synthesized, subunit-based vaccine against tuberculosis.
... and other important pathogens. 28,[35][36][37][38][39] Additionally, the degree of polymorphism in PfRH4 was analyzed in the so identified sequences and their interactions with RBCs were partially characterized by means of saturation, enzymatic treatment, cross-linking, and invasion inhibition assays, together with circular dichroism studies to determine their main structural features. The results indicate that PfRH4 interacts with human RBCs through well-defined regions and support suggesting these regions as excellent candidates to be included in further studies for the design of a multiantigenic, multistage, fully effective, minimal subunit-based synthetic vaccine against P. falciparum malaria. ...
Article
Two widely studied parasite protein families are considered attractive targets for developing a fully effective antimalarial vaccine: the erythrocyte binding antigen (EBA) family defining a sialic acid-dependent invasion pathway, and reticulocyte-binding homologue (RH) proteins associated with sialic acid-independent red blood cell (RBC) invasion. In this study, the micronemal invasive PfRH4 protein was finely mapped using 20-mer-long synthetic peptides spanning the entire protein length to identify protein regions that establish high affinity interactions with human RBCs. Twenty conserved, mainly alpha-helical high-activity binding peptides (HABPs) with nanomolar dissociation constants and recognizing 32, 25, 22, and 20 kDa RBC membrane molecules in a chymotrypsin and/or trypsin-sensitive manner were identified in this protein. Anti-PfRH4 rabbit sera and PfRH4 HABPs inhibited merozoite invasion in vitro, therefore suggesting the implication of these HABPs in Plasmodium falciparum invasion and supporting their inclusion in further structural and immunological studies to design potential components of a minimal subunit-based, multiantigenic, chemically synthesized antimalarial vaccine.
... Nevertheless, it has been also found that specific amino acid replacements made to such peptides (performed according to physicochemical parameters) induce shifts in their tridimensional structure, improving their immunogenicity and protection-inducing ability [30]. Based on such approach, preliminary ligand-receptor interaction studies have been conducted with P. vivax proteins, such as MSP-1, RBP1 and DBP, leading to the identification of peptides binding with high activity to reticulocytes [31][32][33]. It would be therefore also relevant to identify binding regions of MSP10 to host cells and assess the immunogenicity of structurally modified MSP10 binding regions, given that RBC invasion is not effectively blocked nor reduced by antibodies induced by immunization with the unmodified rMSP10. ...
Article
Although largely considered benign, Plasmodium vivax causes disease in nearly 75 million people each year and the available strategies are not sufficient to reduce the burden of disease, therefore pointing to vaccine development as a cost-effective control measure. In this study, the P. vivax merozoite surface protein 10 (MSP-10) was expressed as a recombinant protein in Escherichia coli and purified by affinity chromatography. High antigenicity was observed since sera from P. vivax-infected patients strongly recognized rPvMSP10. The immunogenicity of rPvMSP10 was tested in Aotus monkeys, comparing responses induced by formulations with Freund's adjuvant, Montanide ISA720 or aluminum hydroxide. All formulations produced high antibody titers recognizing the native protein in late schizonts. Despite inducing strong antibody production, none of the formulations protected immunized Aotus monkeys upon experimental challenge.
... In the continuous search for new candidates to be included in a minimal subunit-based, multi-stage, multi-epitopic chemically synthesized vaccine against malaria, our institute has developed a highly robust, specific and sensitive methodology that allows identifying the regions of different pathogen proteins that specifically interact with their corresponding host cell (Mycobacterium tuberculosis, Epstein-Barr virus, Hepatitis C virus, Plasmodium spp., among others) [35][36][37][38][39][40][41]. Herein, we have identified and characterized amino acid sequences derived from the E-TRAMP and REX proteins having high specific binding activity to RBCs. ...
Article
Severe malaria pathology is directly associated with cytoadherence of infected red blood cells (iRBCs) to healthy RBCs and/or endothelial cells occurring during the intraerythrocytic development of Plasmodium falciparum. We synthesized, as 20-mer long peptides, the members of the ring exported (REX) protein family encoded in chromosome 9, as well as the early transcribed membrane proteins (E-TRAMP) 10.2 and 4, to identify specific RBC binding regions in these proteins. Twelve binding peptides were identified (designated as HABPs): three were identified in REX1, two in REX2, one in REX3, two in REX4 and four in E-TRAMP 10.2. The majority of these HABPs was conserved among different P. falciparum strains, according to sequence analysis. No HABPs were found in E-TRAMP 4. Bindings of HABPs were saturable and sensitive to the enzymatic treatment of RBCs and HABPs had different structural features, according to circular dichroism studies. Our results suggest that the REX and E-TRAMP families participate in relevant interactions with RBC membrane proteins, which highlight these proteins as potential targets for the development of fully effective immunoprophylactic methods.
... This method was used for determining specific binding regions in proteins from simple pathogens such as hepatitis C virus , Human papillomavirus (Vera-Bravo et al. 2003), and Epstein-Barr virus (Urquiza et al. 2004). It has also been used in more complex protozoa such as Leishmania (Puentes et al. 1999), Plasmodium falciparum and Plasmodium vivax Rodriguez et al. 2002;Urquiza et al. 2002), malarial blood Ocampo et al. 2005), and hepatic stages Puentes et al. 2004). ...
Article
Virulence and immunity are still poorly understood in Mycobacterium tuberculosis. The H37Rv M. tuberculosis laboratory strain genome has been completely sequenced, and this along with proteomic technology represent powerful tools contributing toward studying the biology of target cell interaction with a facultative bacillus and designing new strategies for controlling tuberculosis. Rv2004c is a putative M. tuberculosis protein that could have specific mycobacterial functions. This study has revealed that the encoding gene is present in all mycobacterium species belonging to the M. tuberculosis complex. Rv2004c gene transcription was observed in all of this complex's strains except Mycobacterium bovis and Mycobacterium microti. Rv2004c protein expression was confirmed by using antibodies able to recognize a 54-kDa molecule by immunoblotting, and its location was detected on the M. tuberculosis surface by transmission electron microscopy, suggesting that it is a mycobacterial surface protein. Binding assays led to recognizing high activity binding peptides (HABP); five HABPs specifically bound to U937 cells, and six specifically bound to A549 cells. HABP circular dichroism suggested that they had an alpha-helical structure. HABP-target cell interaction was determined to be specific and saturable; some of them also displayed greater affinity for A549 cells than U937 cells. The critical amino acids directly involved in their interaction with U937 cells were also determined. Two probable receptor molecules were found on U937 cells and five on A549 for the two HABPs analyzed. These observations have important biological significance for studying bacillus-target cell interactions and implications for developing strategies for controlling this disease.
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Malaria caused by Plasmodium vivax is a neglected disease which is responsible for the highest morbidity in both Americas and Asia. Despite continuous public health efforts to prevent malarial infection, an effective antimalarial vaccine is still urgently needed. P. vivax vaccine development involves analyzing naturally-infected patients' immune response to the specific proteins involved in red blood cell invasion. The P. vivax rhoptry neck protein 2 (PvRON2) is a highly conserved protein which is expressed in late schizont rhoptries; it interacts directly with AMA-1 and might be involved in moving-junction formation. Bioinformatics approaches were used here to select B- and T-cell epitopes. Eleven high-affinity binding peptides were selected using the NetMHCIIpan-3.0 in silico prediction tool; their in vitro binding to HLA-DRB1*0401, HLA-DRB1*0701, HLA-DRB1*1101 or HLA-DRB1*1302 was experimentally assessed. Four peptides (39152 (HLA-DRB1*04 and 11), 39047 (HLA-DRB1*07), 39154 (HLADRB1*13) and universal peptide 39153) evoked a naturally-acquired T-cell immune response in P. vivax-exposed individuals from two endemic areas in Colombia. All four peptides had an SI greater than 2 in proliferation assays; however, only peptides 39154 and 39153 had significant differences compared to the control group. Peptide 39047 was able to significantly stimulate TNF and IL-10 production while 39154 stimulated TNF production. Allele-specific peptides (but not the universal one) were able to stimulate IL-6 production; however, none induced IFN-γ production. The Bepipred 1.0 tool was used for selecting four B-cell epitopes in silico regarding humoral response. Peptide 39041 was the only one recognized by P. vivax-exposed individuals' sera and had significant differences concerning IgG subclasses; an IgG2 > IgG4 profile was observed for this peptide, agreeing with a protection-inducing role against P. falciparum and P. vivax as previously described for antigens such as RESA and MSP2. The bioinformatics results and in vitro evaluation reported here highlighted two T-cell epitopes (39047 and 39154) being recognized by memory cells and a B-cell epitope (39041) identified by P. vivax-exposed individuals' sera which could be used as potential candidates when designing a subunit-based vaccine.
Article
Elucidating receptor-ligand and protein-protein interactions represents an attractive alternative for designing effective Plasmodium vivax control methods. This article describes the ability of P. vivax rhoptry neck proteins 2 and 4 (RON2 and RON4) to bind to human reticulocytes. Biochemical and cellular studies have shown that two PvRON2- and PvRON4-derived conserved regions specifically interact with protein receptors on reticulocytes marked by the CD71 surface transferrin receptor. Mapping each protein fragment's binding region led to defining the specific participation of two 20 amino acid-long regions selectively competing for PvRON2 and PvRON4 binding to reticulocytes. Binary interactions between PvRON2 (ligand) and other parasite proteins, such as PvRON4, PvRON5 and apical membrane antigen 1 (AMA1) were evaluated and characterised by surface plasmon resonance (SPR). The results revealed that both PvRON2 cysteine-rich regions strongly interact with PvAMA1 domains II and III (equilibrium constants in the nanomolar range) and at a lower extent with the complete PvAMA1 ectodomain and domains I and II. These results strongly support that these proteins participate in P. vivax's complex invasion process, thus providing new pertinent targets for blocking P. vivax merozoites' specific entry to their target cells.
Article
Identifying the principles and rules for developing a logical, rational vaccine methodology against various diseases, is discussed. SPf66, the first multiantigenic, multistage, minimal subunit-based, chemically synthesized anti-P. falciparum malaria vaccine. A robust, sensitive, and specific methodology is developed for defining the intimate molecular interactions mediating merozoite invasion of RBC by synthesizing short merozoite-derived protein peptides binding specifically and with high affinity (HABP) to RBCs. Conserved HABP-mediated sporozoite binding to hepatic cells is identified in EBA-175 for developing a multiantigenic, multistage, fully protective antimalarial vaccine. for developing a logical and rational vaccine methodology at the molecular level, monkeys' immune system molecules is analyzed by cloning and sequencing the Aotus genes encoding immunoglobulins, cytokines, and Class I and II molecules.
Article
Receptor-ligand interactions between synthetic peptides and normal human erythrocytes were studied to determine P. falciparum merozoite surface protein-10 (MSP-10) regions specifically binding to membrane surface receptors on human erythrocytes. Three MSP-10 protein High Activity Binding Peptides (HABPs) were identified, whose binding to erythrocytes became saturable and sensitive on being treated with neuraminidase, trypsin and chymotrypsin. Some of them specifically recognised a 50 kDa erythrocyte membrane protein. Some HABPs inhibited in vitro P. falciparum merozoite invasion of erythrocytes by 70%, suggesting that MSP-10 protein's possible role in the invasion process probably functions by using similar mechanisms to those described for other MSP family antigens. In addition to above results, the high homology in amino-acid sequence and superimposition of both MSP-10, MSP-8 and MSP-1 EGF-like domains and HABPs 31132, 26373 and 5501 suggest that tridimensional structure could be playing an important role in the invasion process and in designing synthetic multi-stage anti-malarial vaccines.
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Genome sequencing of the relapsing fever spirochetes Borrelia hermsii and Borrelia turicatae identified three open reading frames (ORFs) on the chromosomes that contained internal, tandemly repeated amino acid sequences that were absent in the Lyme disease spirochete Borrelia burgdorferi. The predicted amino acid sequences of these genes (BH0209, BH0512, and BH0553) have hydrophobic N termini, indicating that these proteins may be secreted. B. hermsii transcribed the three ORFs in vitro, and the BH0512- and BH0553-encoded proteins (PBH-512 and PBH-553) were produced in vitro and in experimentally infected mice. PBH-512 and PBH-553 were on the spirochete's outer surface, and antiserum to these proteins reduced the adherence of B. hermsii to red blood cells. PCR analyses of 28 isolates of B. hermsii and 8 isolates of B. turicatae demonstrated polymorphism in each gene correlated with the number of repeats. Serum samples from relapsing fever patients reacted with recombinant PBH-512 and PBH-553, suggesting that these proteins are produced during human infection. These polymorphic proteins may be involved in the pathogenicity of these relapsing fever spirochetes and provide a mechanism for antigenic heterogeneity within their populations.
Article
Duffy binding protein (DBP) plays a critical role in Plasmodium vivax invasion of human red blood cells. We previously reported a single-chain antibody fragment (scFv) that was specific to P. vivax DBP (PvDBP). However, the stabilization and the half-life of scFvs have not been studied. Here, we investigated the effect of PEGylated scFvs on their biological activity and stability in vitro. SDS-PAGE analysis showed that three clones (SFDBII-12, -58, and -92) were formed as dimers (about 70 kDa) with PEGylation. Clone SFDBII-58 gave the highest yield of PEGylated scFv. Binding analysis using BIAcore between DBP and scFv showed that both SFDBII-12 and -58 were decreased approximately by two folds at the level of binding affinity to DBP after PEGylation. However, the SFDBII-92 clone still showed a relatively high level of binding affinity (KD=1.02 x 10(-7) M). Binding inhibition assay showed that PEGylated scFv was still able to competitively bind the PvDBP and play a critical role in inhibiting the interactions between PvDBP protein expressed on the surface of Cos-7 cells and Duffy receptor on the surface of erythrocytes. When both scFvs and their PEGylated counterparts were exposed to trypsin, scFv was completely degraded only after 24 h, whereas 35% of PEGylated scFvs remained intact, maintaining their stability against the proteolytic attack of trypsin until 72 h. Taken together, these results suggest that the PEGylated scFvs retain their stability against proteolytic enzymes in vivo, with no significant loss in their binding affinity to target antigen, DBP.
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The malarial GBP 130 protein binds weakly to intact human erythrocytes; the binding sites seem to be located in the repeat region and this region's antibodies block the merozoite invasion. A peptide from this region (residues from 701 to 720) which binds to human erythrocytes was identified. This peptide named 2220 did not bind to sialic acid; the binding site on human erythrocyte was affected by treatment with trypsin but not by chymotrypsin. The peptide was able to inhibit Plasmodium falciparum merozoite invasion of erythrocytes. The residues F701, K703, L705, T706, E713 (FYKILTNTDPNDEVERDNAD) were found to be critical for peptide binding to erythrocytes.
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A novel yet simple method is described that facilitates the synthesis of large numbers of peptides to the extent that the synthesis process need no longer be the limiting factor in many studies involving peptides. By using the methods described, 10-20 mg of 248 different 13-residue peptides representing single amino acid variants of a segment of the hemagglutinin protein (HA1) have been prepared and characterized in less than 4 weeks. Through examination of the binding of these analogs to monoclonal antibodies raised against residues 75-110 of HA1, it was found that a single amino acid, aspartic acid at position 101, is of unique importance to the interaction. Two other residues, aspartic acid-104 and alanine-106, were found to play a lesser but significant role in the binding interaction. Other single positional residue variations appear to be of little or no importance.
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A short-term in vitro culture system that allows for significant re-invasion of target erythrocytes by Plasmodium vivax was used to study the role of the Duffy blood group antigen as a ligand for merozoite invasion by this human malaria species. Using human Duffy-positive and -negative erythrocytes, various primate erythrocytes, enzymatic modification of erythrocytes, and mAb that defines a new Duffy determinant (Fy6) we conclude that the erythrocyte glycoprotein carrying Duffy determinants is required as a ligand for the invasion of human erythrocytes by P. vivax merozoites. Blockade of invasion by Fab fragments of the anti-Fy6 mAb equal to that of the intact molecule and the correlation of P. vivax susceptibility with the presence of the Fy6 determinant suggests this epitope or a nearby domain may be an active site on the Duffy glycoprotein. However, as for P. knowlesi, there is evidence that an alternate pathway for P. vivax invasion of simian erythrocytes may exist.
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The erythrocyte chemokine receptor is a cell surface protein that binds a wide array of chemokines including interleukin-8 (IL-8), melanoma growth stimulating activity (MGSA), monocyte chemotactic protein-1 (MCP-1), and RANTES (Regulated on Activation, Normal T Expressed and Secreted). This protein has also been identified as the Duffy blood group antigen, a cell surface receptor for the malarial parasite Plasmodium vivax. In the present study, we have identified a chemokine receptor-like binding protein in a human erythroleukemic cell line (HEL), which, based on its molecular properties, may be related to the erythrocyte chemokine receptor. Saturation binding studies with 125I-IL-8 revealed a single class of IL-8 binding sites in HEL cells with a KD of 7.4 +/- 1.9 nM and a receptor density of 12,818 +/- 965 binding sites/cell. In competition studies unlabeled IL-8 MGSA, MCP-1, and RANTES were fully able to inhibit the binding of 125I-IL-8 to HEL cells. Chemical cross-linking with radiolabeled IL-8 resulted in a cross-linked species of 60 kDa in membranes from HEL cells. The labeling was specific since it was inhibited by pre-incubation with 1 microM unlabeled IL-8 or MGSA. A monoclonal antibody (Fy6) to the human erythrocyte Duffy blood group antigen/chemokine receptor blocked the binding of IL-8 and other chemokines to the HEL cell chemokine receptor-like binding protein. Cell membranes from HEL cells and from erythrocyte ghosts were subjected to SDS-PAGE and analyzed by Western blotting with anti-Fy6. The antibody bound to a molecule with a molecular mass of 50 kDa in HEL cell membranes and 40 kDa in erythrocyte ghosts. Northern blot analysis of mRNA revealed that the HEL chemokine-binding protein hybridized to a cDNA probe to the Duffy antigen/chemokine receptor.
Article
Solid experimental evidence indicates that EBA-175 is used as a ligand by the Plasmodium falciparum merozoite to bind to human RBC, via different binding processing fragments. Using synthetic peptides and specific receptor-ligand interaction methodology, we have identified 6 high-activity binding sequences from the EBA-175 CAMP strain; peptide 1758 (KSYGTPDNIDKNMSLIHKHN), located in the so-called region I for which no binding activity has been reported before, peptides 1779 (NIDRIYDKNLLMIKEHILAI) and 1783 (HRNKKNDKLYRDEWWKVIKK), located in region II, in a sub-region known as 5' Cys F2, previously reported as being a binding region, and peptides 1814 (DRNSNTLHLKDPRNEENERH), 1815 (YTNQNINISQERDLQKHGFH) and 1818 (NNNFNNIPSRYNLYDKKLDL), in region III-V where antibodies inhibit merozoite invasion of erythrocytes. The affinity constants were between 60 and 180 nM and the critical amino acids involved in the binding were identified. The binding of these peptides to enzyme-treated RBC was analysed; binding of peptide 1814, located in the III-V region, was found to be sialic acid dependent. Some of these high binding peptides were able to inhibit in vitro merozoite invasion and to block the binding of recombinant RII-EBA ro RBC. Several of these peptides are located in regions recognized by protective immune clusters of merozoites (ICMs) eluted antibodies.
Article
An SN2 deprotection reaction for synthetic peptides was observed when the weak base dimethyl sulfide was used as a diluent for HF. Kinetic studies of the deprotection of O-benzylserine revealed that there was a sharp changeover in mechanism from AAL1 to AAL2 when the concentration of HF in dimethyl sulfide was below 55%. The changeover in mechanism was also found in the deprotection of O-benzyltyrosine. At higher HF concentrations (>55%), the AAL1 cleavage mechanism, which generates carbonium ions, led to significant 3-benzyltyrosine side product. However, at low HF concentrations, the side product was minimal as a result of an AAL2 cleavage mechanism in which carbonium ions are not formed. A sharp increase of side product was seen when the HF concentration reached the critical changeover concentration. The HF-dimethyl sulfide reagent was also found to reduce methionine sulfoxide to methionine and, in the presence of a thiol, to deprotect Ni-formyltryptophan to tryptophan. Both of these reactions were also dependent on the concentration of HF and were optimal at low concentrations. Furthermore, deprotection of aspartic and glutamic acid side chain benzyl esters at the low HF concentration also minimized the AAC1 mechanism and the accompanying acylation side reactions. A practical mixture for the SN2 deprotection reaction was found to be HF-dimethyl sulfide-p-cresol (25:65:10 v/v). For the deprotection of Trp(For)-containing peptides, the reagent was adjusted to HF-dimethyl sulfide-p-cresol-p-thiocresol (25:65:7.5:2.5 v/v) so that the Ni-formyl could be removed concomitantly with other protecting groups. The low-acidity function, SN2 reaction was also effective for solid-phase peptide synthesis. The same protecting groups were removed as in solution, and in addition the bond holding the peptide to the resin support was cleaved. For more resistant anchoring bonds and protecting groups a combined low-high HF procedure was developed, in which most of the precursors of harmful carbonium ions are removed by a SN2 mechanism before the final strong-acid, SN1, step begins. The new deprotection procedure was tested on three synthetic model peptides, methionine-enkephalin, bovine growth hormone fragment (128-131), and C-terminal pentagastrin amide, and was found to provide efficient deprotection and significant reduction in the level of alkylation side reactions, the rearrangement to aspartimide, and the acylation of aromatic scavengers by glutamic acid.
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A new approach to the chemical synthesis of polypeptides was investigated. It involved the stepwise addition of protected amino acids to a growing peptide chain which was bound by a covalent bond to a solid resin particle. This provided a procedure whereby reagents and by-products were removed by filtration, and the recrystallization of intermediates was eliminated. The advantages of the new method were speed and simplicity of operation. The feasibility of the idea was demonstrated by the synthesis of the model tetrapeptide L-leucyl-L-alanylglycyl-L-valine. The peptide was identical with a sample prepared by the standard p-nitrophenyl ester procedure.
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The role of AMA-1 during merozoite invasion has not yet been determined. However, reported experimental evidence suggests that this protein can be used, in particular as erythrocyte-binding protein, since, Fab fragments against this protein are able to block merozoite invasion. Using a previously described methodology, eight peptides with high binding activity to human erythrocyte, scattered along the different domains and having around 130 nM affinity constants, were identified in the Plasmodium falciparum AMA-1 protein. Their binding activity was sialic acid independent. Some of these peptides showed homology with the erythrocyte binding domains of one of the apical organelle protein family, MAEBL, identified in rodent malarial parasites. One of these peptides shares amino acid sequence with a previously reported B-cell epitope which induces antibodies to block parasite growth. The critical residues were identified for erythrocyte binding conserved peptides 4313 (DAEVAGTQPSGKCPVFG), 4321 (VVDNKVPRKNQNAKFG), 4325 (MIKFLPTGAFKRKSH) and 4337 (GEKRASTTPVLMEKPYY). All conserved peptides were able to block merozoite invasion of new RBC and development, suggesting that these peptides are involved in P. falciparum invasion.
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Article
Plasmodium vivax merozoites primarily invade reticulocytes. The basis of this restricted host cell preference has been debated. Here we introduce two novel P. vivax proteins that comigrate on reducing SDS-polyacrylamide gels, colocalize at the apical pole of merozoites, and adhere specifically to reticulocytes. The genes encoding these proteins, P. vivax reticulocyte-binding proteins 1 and 2 (PvRBP-1 and PvRBP-2), have been cloned and analyzed. Homologous genes are evident in the closely related simian malaria parasite, P. cynomolgi, which also prefers to invade reticulocytes, but are not evident in the genome of another related simian malaria parasite, P. knowlesi, which invades all red blood cell subpopulations. Native PvRBP-1 is likely a transmembrane-anchored disulfide-linked protein, and along with PvRBP-2 may function as an adhesive protein complex. We propose that the RBPs of P. vivax, and homologous proteins of P. cynomolgi, function to target the reticulocyte subpopulation of red blood cells for invasion.
Article
The human malaria parasite Plasmodium falciparum demonstrates variability in its dependence upon erythrocyte sialic acid residues for invasion. Some lines of P. falciparum invade neuraminidase-treated or glycophorin-deficient red blood cells poorly, or not at all, while other lines invade such cells at substantial rates. To explore the molecular basis of non-sialic acid dependent invasion, we selected parasite lines from a clone (Dd2) that initially exhibited low invasion of neuraminidase-treated erythrocytes. After maintaining Dd2 for several cycles in neuraminidase-treated erythrocytes, parasite lines were recovered that invaded both untreated and neuraminidase-treated erythrocytes at equivalently high rates (Dd2/NM). The change in phenotype was maintained after removal of selection pressure. Four subclones of Dd2 were isolated and each readily converted from sialic acid dependence to non-sialic acid dependence during continuous propagation in neuraminidase-treated erythrocytes. The neuraminidase-selected lines and the Dd2 clone demonstrated identical restriction fragment length polymorphism markers indicating that the Dd2 clone was not contaminated during the selection process. Parasite proteins that bound to neuraminidase-treated and untreated erythrocytes were indistinguishable among the parent Dd2 clone and the neuraminidase-selected lines. The ability of the Dd2 parasite to change its invasion requirements for erythrocyte sialic acid suggests a switch mechanism permitting invasion by alternative pathways.
Article
N-Acetylneuraminic acid (NeuNAc) is the terminal sugar residue of the O-linked tetrasaccharide linked to erythrocyte sialoglycoproteins, glycophorins. Erythrocytes lacking NeuNAc have been shown previously to be resistant to invasion by certain isolates of Plasmodium falciparum merozoites. We report here variation between different geographic isolates of P. falciparum in their dependency on NeuNAc for invasion of host erythrocytes. Seven different geographic isolates of P. falciparum were examined for their ability to invade neuraminidase treated erythrocytes. For all isolates invasion was reduced significantly, although considerable variation in NeuNAc dependency was apparent. Three isolates, FCR-3, FVO and It2, exhibited a very high dependence on NeuNAc residues for invasion (invasion reduced greater than 90%), whereas two isolates (Thai-Tn and FC-27) exhibited a moderately high dependence (invasion reduced 75%). Two other isolates (CDC-1 and 7G8) exhibited moderate dependence on NeuNAc (invasion reduced 50%). Cleavage of the complete O-linked tetrasaccharide by O-glycanase removes all carbohydrate from glycophorin A, B and C except the single N-linked oligosaccharide on glycophorin A and C. Invasion of FCR-3 and CDC-1 isolates into O-glycanase treated erythrocytes was not markedly different from that into neuraminidase treated cells indicating that NeuNAc is the important residue of the tetrasaccharide for both isolates. Invasion into endo-beta-galactosidase treated erythrocytes, in which the lactosaminoglycan side chain of band 3 and band 4.5 is cleaved, was not significantly reduced for either the CDC-1 or FCR-3 isolates. Additional results on the trypsin insensitivity of band 3 also suggest that this erythrocyte protein is not important in P. falciparum recognition. The greatest divergence in receptor specificity between FCR-3 and CDC-1 isolates was apparent in invasion into periodate-treated erythrocytes. Periodate oxidation results in cleavage of the exocyclic hydroxyl groups of the terminal NeuNAc but leaves its COOH group unaltered. These experiments also illustrated that the negatively charged COOH group of NeuNAc is not the important group in the interaction of the merozoite with the NeuNAc. Trypsin-treated erythrocytes were almost fully resistant to invasion by CDC-1 as well as the FCR-3 isolates suggesting that the CDC-1 isolate, in addition to interacting with NeuNAc, depends on a trypsin sensitive site for invasion. This site could involve the N-linked saccharide on glycophorin A and C or a protein on the erythrocyte surface unrelated to the glycophorins.
Article
Studies on the morphology, cell biology, and immunology of invasion have characterized events that are now being studied at the molecular level. The initial events of invasion are receptor-specific. A determinant associated with Duffy blood group antigens is involved in the invasion of human erythrocytes by P. knowlesi and P. vivax. The Duffy Fya antigen has recently been identified and further characterization of its role in reception and invasion should now be possible. P. falciparum utilizes erythrocyte ligands that differ from those of P. knowlesi and P. vivax. Sialic acid and a trypsin-sensitive erythrocyte membrane component are important for invasion by P. falciparum parasites. There is evidence that at least two ligands are involved in invasion. For P. knowlesi there is a ligand for attachment, common to both Duffy-negative and Duffy-positive human erythrocytes, and a second ligand for invasion, which is found only on Duffy-positive human erythrocytes. P. vivax also appears to utilize two ligands, a Duffy-associated ligand and a ligand specific for reticulocytes. P. falciparum binds to sialic acid-dependent and sialic acid-independent trypsin-sensitive ligands. P. falciparum merozoites require erythrocyte sialic acid to varying degrees in order to invade; this indicates heterogeneity of the receptor mechanism. Monoclonal antibodies and recombinant DNA technology have greatly facilitated the identification, isolation, and characterization of proteins that may be involved in invasion. Molecules that may have invasion-related functions include those whose antibodies block invasion, those that bind to erythrocyte ligands important for invasion, those that appear on the merozoite surface, and those that appear to be inserted into the erythrocyte membrane at the time of invasion. It has not been possible to identify a definite function for any of the molecules identified thus far. No monoclonal or polyclonal monospecific antibody has been identified that reacts specifically over the surface of the apical region of the merozoite where junction formation occurs. Identification of molecules responsible for apical attachment and junction formation will be important for our understanding of invasion. In terms of vaccine development, it is not yet known whether any of the molecules discussed here will prove to be effective immunogens. It is clear from the data obtained with the 140-kd protein of P. knowlesi that antigenic variation poses a potential problem for vaccine development. As the molecular events responsible for invasion become better understood, novel ways may be devised to interfere with the process and prevent the disease.
Article
Antigens that bind to erythrocytes were identified in the supernatant fluids of a cultured human malaria parasite (Plasmodium falciparum). A 175-kilodalton (175K) antigen bound only to erythrocytes susceptible to invasion. The 175K antigen from the Camp or the FCR-3 strain also bound to merozoites. However, the antigen did not bind to merozoites when merozoites and supernatant antigens were from different strains unless proteinase inhibitors were present. Moreover, erythrocytes coated with supernatant antigens from the Camp or FCR-3 strain were invaded normally by merozoites of the homologous strain but were partially resistant to invasion by merozoites of the heterologous strain. The 175K antigen may be a receptor acting as a "bridge" between erythrocytes and merozoites.
Article
It has recently become possible to characterize a variety of different receptors by studying the binding of appropriate drugs labelled with 3H or 125I. The goal of this review is to describe the basic mathematical analyses that should be used to characterize a particular receptor in terms of its interactions with ligands. Methods for direct determination of kinetic and equilibrium constants of simple bimolecular drug-receptor interactions are described as is the use of these measurements to verify the existence of a simple second order reaction. Some of the causes of deviations from second order behavior which imply more complex interactions are also discussed. studies of radioligand binding provide a means of indirectly determining equilibrium dissociation constants of unlabelled drugs. The appropriate equations for these determinations are presented and the assumptions underlying these calculations are identified. Analysis of the temperature dependence of kinetic and equilibrium constants allows determination of the energetics of binding and methods are presented for calculation of the changes in Gibbs free energy, enthalpy and entropy that are associated with the binding of ligands to receptors. Studies of the interactions of agonists and antagonists with β-adrenergic receptors are presented as examples of the various types of calculations.
Article
Invasion of erythrocytes by malaria parasites involves multiple receptor-ligand interactions. To elucidate these pathways, we made use of four parasite clones with differing specificities for invasion, erythrocytes that are mutant for either glycophorin A or B, and enzyme modification of the erythrocyte surface with neuraminidase and trypsin. Neuraminidase alone abolishes invasion of two parasite clones (Dd2, FCR3/A2); these invade after trypsin treatment alone. A third clone (7G8) is unable to invade trypsin-treated erythrocytes. The fourth clone (HB3) can invade after either neuraminidase or trypsin treatment. The receptor for invasion of trypsin-treated erythrocytes was explored in two ways: treatment of trypsin-treated normal cells with neuraminidase, and trypsin treatment of glycophorin B-deficient cells. Both treatments eliminated invasion by all clones, indicating that the trypsin-independent pathway uses sialic acid and glycophorin B. To identify parasite proteins involved in the different pathways, erythrocyte binding assays were performed with soluble parasite proteins from each clone. Based on binding assays using erythrocytes that lack glycophorin A, the parasite protein known as EBA-175 appears to bind predominantly to glycophorin A. In contrast, the glycophorin B pathway does not appear to involve EBA-175, as binding of EBA-175 was similarly reduced to trypsin-treated normal and trypsin-treated glycophorin B-deficient erythrocytes. Thus, the glycophorin B-dependent, sialic acid-dependent invasion of trypsin-treated normal erythrocytes uses a different parasite ligand, indicating two or more sialic-dependent pathways for invasion. Clone 7G8, which cannot invade trypsin-treated erythrocytes, may be missing the ligand for invasion via glycophorin B.(ABSTRACT TRUNCATED AT 250 WORDS)
Article
To determine amino acid sequences of the Plasmodium falciparum MSP-1 protein that interact with red blood cell membranes in a specific receptor-ligand interaction, 78 sequential peptides, 20 amino acids long and spanning the entire length of the molecule, were synthesized and analysed with a specific binding assay developed for this purpose. Results show that peptides based on conserved and dimorphic regions of MSP-1, interact with human red blood cells (RBCs). This interaction occurs predominantly with peptides contained within the MSP-1 proteolytic fragments of 83 kDa, 38 kDa, 33 kDa and 19 kDa. Affinity constants of these peptides were between 140 and 250 nM. Peptide-RBC binding post enzyme treatment showed that the RBC receptors are not sialic acid dependent and appear to be proteic in nature. Some of these peptides inhibited merozoite invasion of RBCs yet did not inhibit intraerthrocytic development. These peptides, in conjunction with those from other merozoite surface proteins, may be used to rationally design a second generation of synthetic peptide-based malaria vaccines.
Article
The Ring-Infected Erythrocyte Surface Antigen (Pf155/RESA) sequence was chemically synthesized in fifty four 20-mer sequential peptides, covering the entire protein, each of which was tested in erythrocyte binding assays. Peptides 6671 and 6673, corresponding to residues 141-160 and 181-200, respectively, presented a high specific binding activity to erythrocytes with affinity constants of 190 nM and 105 nM respectively. Their binding was sensitive to previous enzymatic treatment of erythrocytes. A region of peptide 6673 has been identified, very recently, as a B-cell epitope, target of neutralizing antibodies (Siddique AB, Iqbal J, Ahlborg N, Wâhlin FB, Perlmann P, Berzins K. Antibodies to nonrepeat sequences of antigen Pf155/RESA of Plasmodium falciparum inhibit parasite growth in vitro. Parasitol Res 1998;84:485-91). The critical residues for erythrocyte binding for peptide 6671 (MTDVNRYRYSNNYEAIPHIS) and for peptide 6673 (LGRSGGDIIKKMQTLWDEIM) were recognized. Based on these data, the presence of five functional regions of RESA is postulated.
Article
Plasmodium falciparum merozoite membrane surface antigen 2 (MSA2) has been associated with the development of protective immunity against malaria. MSA2 antibodies were able to inhibit in vitro merozoite invasion. In our search for experimental evidence concerning the participation of MSA2 in merozoite invasion, 40 peptides were synthesized according to sequences reported for the CAMP and FC27 prototype Plasmodium strains. These peptides were purified, 125I-radiolabeled and tested for their ability to bind to erythrocytes. Two MSA2 synthetic peptides with high specific binding to human erythrocytes were found. The peptide coded 4044 (KNESKYSNTFINNAYNMSIR), located in the MSA2 N-terminal conserved region, has an affinity coefficient of 72 nM and showed a positive cooperativity for the receptor-ligand interaction. The other peptide, coded 4053 (NPNHKNAETNPKGKGEVQKP) and located in the central variable region of MSA2, has an affinity coefficient of 49nM and also showed a positive cooperativity for the receptor-ligand interaction. The binding capacity of these peptides is affected by erythrocytes treated with neuraminidase and trypsin, but it is not affected by chymotrypsin. Both of these sequences inhibit in vitro erythrocyte parasite invasion by up to 95% suggesting that they have an important role in the parasite's invasion process. Furthermore, as published previously [A. Saul et al. (1992) J. Immunol., 148, 208-211], a protective B epitope is included in the 4044 peptide sequence.
Article
The Plasmodium vivax merozoite surface protein-1 (PvMSP-1) has been considered a candidate for a malaria vaccine against erythrocytic stages. PvMSP-1 is immunogenic during natural infections and exhibits antigenic polymorphism. The extent of genetic polymorphism in a region between the so-called interspecies conserved blocks (ICBs) 2 and 4 of the PvMSP-1 was analyzed in 20 isolates taken from patients from two different areas in Colombia. Variation is unevenly distributed along this gene segment among the isolates. Comparative analysis of these sequences led to the definition of five sequence types (ST1 to 5). ST1 to ST4 exhibit a variation pattern associated with sequences present in the Salvador or Belem sequences. However, ST5 has clusters of sequence that have not been previously described. The changes found along the five variants confirm the important role of recombinational and/or gene conversion events in generating allelic diversity.
Article
Plasmodium vivax Duffy Binding Protein (Pv-DBP) is essential during merozoite invasion of reticulocytes. Reticulocyte binding region identification is important for understanding Pv-DBP reticulocyte recognition. Fifty 20 mer non-overlapping peptides, spanning Pv-DBP sequences, were tested in erythrocyte and reticulocyte binding assays. Ten HARBPs, mainly located in region II (Kd 50-130 nM), were High Activity Reticulocyte Binding Peptides (HARBPs); one bound to erythrocytes. Reticulocyte trypsin-, chymotrypsin- or neuraminidase- treatment affects HARBP binding differently, suggesting that these peptides have different reticulocyte-binding-sites. Some peptides bound to a Coomasie non-stainable 40 Kda band. Some HARBPs were able to block recombinant PvRII binding (Pv-DBP region II) to Duffy positive reticulocytes.
Article
Plasmodium vivax merozoites have high preferential ability to interact with and invade reticulocytes, although these cells correspond to only 2% of the red blood cells (RBC) population. P. vivax merozoite surface protein-1 (Pv-MSP-1) is believed to have an important role in attachment and invasion process. Using 88 non-overlapping 20-mer peptides, covering the entire Pv-MSP-1 Belem strain sequence, RBC and reticulocyte binding assays were performed. Fourteen sequences were identified with high specific binding activity to reticulocytes, but only three had high specific binding activity to mature erythrocytes. These peptides showed affinity constant values between 20 and 150nM, indicating a strong interaction between these sequences and reticulocyte receptors. Critical residues in binding to reticulocytes for these peptides were determined by competition binding assays with glycine scanning analogues. All high binding peptides bind to reticulocyte surface proteins having a molecular mass of around 18-20kDa which are not present in mature RBC. Interestingly, some high activity binding peptides (HABPs) are located close to the hypothesised 42 and 19kDa fragment cleavage sites for this protein, suggesting that these sequences have an important role in target cell attachment and invasion process by Pv-MSP-1.HABPs may be clustered in two regions, with region I being located between amino acids 280-719, and region II between amino acids 1060-1599 with higher than 25% identity level. A P. falciparum MSP-1 antigenic domain binds to RBCs and inhibits parasite invasion. Peptides 1721 and 1724 bind with high activity to reticulocytes in homologous Pv-MSP-1, suggesting similar functions for these two sequences.
Article
Identify hepatitis C virus (HCV) sequences in E1 and E2 protein binding to HepG2. Synthetic 20-mer long, ten-residue overlapped peptides, from E1 and E2 proteins, were tested in HepG2 or Raji cell-binding assays. Affinity constants, binding site number per cell and Hill coefficients were determined by saturation assay for high activity binding peptides (HABPs). Receptors for HepG2 cell were determined by cross-linking and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Twelve HABPs were found in HCV genotype 1a, allowing six hepatocyte-binding sequences (HBSs) to be defined: two peptide-binding regions in E1 HABPs 4913 (YQVRNSTGLYHVTNDCPNSS) and 4918 (MTPTVATRDGKLPATQLRRHY). Four hepatocyte-binding regions were defined in E2: region-I, peptide 4931 (ETHVTGGSAGHTVSGFVSLLY); region-II, 4937-4939 (HHKFNSSGCPERLASCRPLTDFDQGWGPISYANGSGPDQR); region-III, 4943-4945 (PVYCFTPSPVVVGTTDRSGAPTYSWGENDTDVFVLNNTR) and region-IV, 4949-4952 (CGAPPCVIGGAGNNTLHCPTDCFRKHPDATYSRCGSGPWITPRCLVDYPY). The underlined sequences are most relevant in the binding process. HABPs 4913 and 4938 also bind to CD81 positive Raji cells. Region-II 4938 HABPs bind to 50 and 60kDa HepG2 cell membrane surface proteins. Six HVRs to the HepG2 were identified. Some HABPs have been previously found to be antigenic and immunogenic. HABPs, 4918 (from E1), 4938, 4949, 4950, 4951 and 4952 (from E2) have not been previously recognised. These HABPs could be relevant to HCV invasion of hepatocytes.
Article
Several Plasmodium vivax merozoite proteins have been characterized over the past few years, including two that bind specifically to reticulocytes. Here, Mare Galinski and John Barnwell examine P. vivax merozoites and constituent molecules that are involved in host cell selection and invasion, and that also are viewed as malaria vaccine candidates. They also discuss how knowledge of the reticulocyte-binding proteins furthers the development of a conceptual framework for malaria merozoite invasion at the molecular level, not only for P. vivax, but for all species of the parasite.
Article
Resistance of Plasmodium falciparum to chloroquine has emerged in the late 1950s and has now conquered the large majority of areas where this species is endemic. Resistance to alternative drugs has already occurred in several parts of the world and has become a particularly serious problem in Thailand. Emergence and spread of resistance are the result of interactions between parasite, humans, vector and drugs, enhanced by particular ecological features. The control of malaria transmission by means other than drugs would probably curb the propagation of resistance but current health care policies offer only limited prospects for the reactivation or implementation of systematic malaria control before the potential of the affordable antimalarials has been exhausted. In this article, Walther Wernsdorfer considers the epidemiological factors associated with the development and spread of drug-resistant malaria.
Neurotransmitter receptor binding
  • Yamamura Hi
  • Enna
  • Sj
  • Kuhar
  • Mj
Yamamura HI, Enna SJ, Kuhar MJ. Neurotransmitter receptor binding. New York: Raven Press; 1978.
Current protocols in molecular biology
  • Ausubel
  • Brent R Fm
Ausubel FM, Brent R, et al. Current protocols in molecular biology, 2nd ed. New York: Greene Publishing Associates and Wiley Interscience; 1993.