Red Blood Cell Invasion by Plasmodium vivax: Structural Basis for DBP Engagement of DARC

Plasmodium parasites use specialized ligands which bind to red blood cell (RBC) receptors during invasion. Defining the mechanism of receptor recognition is essential for the design of interventions against malaria. Here, we present the structural basis for Duffy antigen (DARC) engagement by P. vivax Duffy binding protein (DBP). We used NMR to map the core region of the DARC ectodomain contacted by the receptor binding domain of DBP (DBP-RII) and solved two distinct crystal structures of DBP-RII bound to this core region of DARC. Isothermal titration calorimetry studies show these structures are part of a multi-step binding pathway, and individual point mutations of residues contacting DARC result in a complete loss of RBC binding by DBP-RII. Two DBP-RII molecules sandwich either one or two DARC ectodomains, creating distinct heterotrimeric and heterotetrameric architectures. The DARC N-terminus forms an amphipathic helix upon DBP-RII binding. The studies reveal a receptor binding pocket in DBP and critical contacts in DARC, reveal novel targets for intervention, and suggest that targeting the critical DARC binding sites will lead to potent disruption of RBC engagement as complex assembly is dependent on DARC binding. These results allow for models to examine inter-species infection barriers, Plasmodium immune evasion mechanisms, P. knowlesi receptor-ligand specificity, and mechanisms of naturally acquired P. vivax immunity. The step-wise binding model identifies a possible mechanism by which signaling pathways could be activated during invasion. It is anticipated that the structural basis of DBP host-cell engagement will enable development of rational therapeutics targeting this interaction.     

Purification and characterization of an erythrocyte membrane protein complex carrying Duffy blood group antigenicity. Possible receptor for Plasmodium vivax and Plasmodium knowlesi malaria parasite

A murine monoclonal antibody, named anti-Fy6, which agglutinates all human red cells except those of Fy(a-b) phenotype was used for purification and characterization of Duffy antigens. Duffy antigens are multimeric red cell membrane proteins composed of different subunits of which only one, designated pD protein, reacts in immunoblots with the murine monoclonal antibody anti-Fy6. Affinity-purified detergent-soluble antigen-antibody complex obtained from red cells, surface-labeled with 125I yielded a complex pattern of bands when separated by polyacrylamide gel electrophoresis. Proteins that react with anti-Fy6 in immunoblots are: pA and pB (greater than 100 kDa) and pD (36-46 kDa). Electroeluted pD protein aggregates and generates bands of similar molecular mass to pA and pB proteins. Electroeluted pA and pB proteins disaggregate yielding pD protein. Oligomers and monomers of pD protein are present in red cells carrying Duffy antigens and absent in Fy(a-b-) cells. Six other proteins of molecular weight ranging from 68 to 21 kDa either associate or co-purify with pD protein. These proteins are only present in Duffy antigen positive cells. The pD protein is different in Fy(a+b-) and Fy(a-b+) cells by fingerprint analysis. Human antisera identify the same proteins in red cell carrying Duffy antigens as the murine monoclonal antibody anti-Fy6.     

A merozoite receptor protein from Plasmodium knowlesi is highly conserved and distributed throughout Plasmodium

The 66-kDa merozoite surface antigen (PK66) of Plasmodium knowlesi, a simian malaria, possesses vaccine-related properties that are thought to originate from a receptor-like role in parasite invasion of erythrocytes. We report the complete sequence of PK66 which allowed the demonstration that highly conserved analogues exist throughout Plasmodium including a recently reported gene from P. falciparum (Peterson, M. G., Marshall, V. M., Smythe, J. A., Crewther, P. E., Lew, A., Silva, A., Anders, R. F., and Kemp, D. J. (1989) Mol. Cell. Biol. 9, 3151-3155). These analogues are highly promising vaccination candidates. The distribution of PK66 changes after schizont rupture in a coordinate manner associated with merozoite invasion. The protein is concentrated at the apical end prior to rupture, following which it can distribute itself entirely across the surface of the free merozoite. During invasion, immunofluorescence studies suggest that, PK66 is excluded from the erythrocyte at, and behind, the invasion interface.     

Plasmodium vivax, P. cynomolgi, and P. knowlesi: identification of homologue proteins associated with the surface of merozoites

We have identified a Plasmodium vivax merozoite surface protein (MSP) that migrates on SDS-polyacrylamide gels at a Mr of about 185 kDa. This protein was recognized by a P. vivax monoclonal antibody (mAb) that localizes the protein by immunofluorescence to the surface of merozoites and also immunoprecipitates this protein from NP-40 detergent extracts of [35S]methionine metabolically radiolabeled P. vivax schizonts. The P. vivax MSP does not become biosynthetically radiolabeled with [3H]glucoamine, [3H]myristate, [3H]palmitate, or [3H]mannose, indicating that this P. vivax MSP is not posttranslationally modified and bound to the merozoite membrane by a glycosylphosphatidylinositol (GPI) lipid anchor. Thus, in this respect, this protein is different from members of the MSP-1 protein family and from MSP-2 and MSP-4 of P. falciparum. The mAb cross-reacts with and outlines the surface of P. cynomolgi merozoites and immunoprecipitates a 150-kDa P. cynomolgi homologue. The mAb was used as an affinity reagent to purify the native homologous MSP from NP-40 extracts of P. cynomolgi mature schizonts in order to develop a specific polyclonal antiserum. The resulting anti-PcyMSP rabbit antiserum cross-reacts strongly with the P. vivax 185-kDa MSP and also recognizes an analogous 110-kDa protein from P. knowlesi. We have determined via an immunodepletion experiment that the 110-kDa P. knowlesi MSP corresponds to the PK 110 protein partially characterized earlier (Perler et al. 1987). The potential of P. vivax MSP as a vaccine candidate was addressed by conducting in vitro inhibition of erythrocyte invasion assays, and the IgG fraction of both the P. vivax MSP mAb and the P. cynomolgi MSP rabbit antiserum significantly inhibited entry of P. vivax merozoites. We denote, on a preliminary basis, these antigenically related merozite surface proteins PvMSP-185, PcyMSP-150, and PkMSP-110.     

Acquired Antibody Responses against Plasmodium vivax Infection Vary with Host Genotype for Duffy Antigen Receptor for Chemokines (DARC)

Background

Polymorphism of the Duffy Antigen Receptor for Chemokines (DARC) is associated with susceptibility to and the severity of Plasmodium vivax malaria in humans. P. vivax uses DARC to invade erythrocytes. Individuals lacking DARC are ‘resistant’ to P. vivax erythrocytic infection. However, susceptibility to P. vivax in DARC+ individuals is reported to vary between specific DARC genotypes. We hypothesized that the natural acquisition of antibodies to P. vivax blood stages may vary with the host genotype and the level of DARC expression. Furthermore, high parasitemia has been reported to effect the acquisition of immunity against pre-erythrocytic parasites. We investigated the correlation between host DARC genotypes and the frequency and magnitude of antibodies against P. vivax erythrocytic stage antigens.

Methodology/Findings

We assessed the frequencies and magnitudes of antibody responses against P. vivax and P. falciparum sporozoite and erythrocytic antigens in Colombian donors from malaria-endemic regions. The frequency and level of naturally-acquired antibodies against the P. vivax erythrocytic antigens merozoite surface protein 1 (PvMSP1) and Duffy binding protein (PvDBP) varied with the host DARC genotypes. Donors with one negative allele (FY*B/FY*Bnull and FY*A/FY*Bnull) were more likely to have anti-PvMSP1 and anti-PvDBP antibodies than those with two positive alleles (FY*B/FY*B and FY*A/FY*B). The lower IgG3 and IgG1 components of the total IgG response may account for the decreased responses to P. vivax erythrocytic antigens with FY*A/FY*B and FY*B/FY*B genotypes. No such association was detected with P. falciparum erythrocytic antigens, which does not use DARC for erythrocyte invasion.

Conclusion/Significance

Individuals with higher DARC expression, which is associated with higher susceptibility to P. vivax infection, exhibited low frequencies and magnitudes of P. vivax blood-stage specific antibody responses. This may indicate that one of the primary mechanisms by which P. vivax evades host immunity is through DARC indirectly down-regulating humoral responses against erythrocytic invasion and development.     

Cultivation of Plasmodium vivax

Establishment of a continuous line of Plasmodium vivax parasite is crucial to understand the parasite's biology; however, this has not yet been achieved. Beginning in the 19th century, there were several efforts to cultivate this malaria parasite but without much success until the late 1980s. In addition, to date, only minor modifications of the methodology have been investigated, which has resulted in extending the cultivation period to around four weeks by supplying reticulocytes obtained from normal blood or rare hemochromatotic blood. However, the use of laboratory-produced erythroblasts to cultivate P. vivax enables maintenance of a continuous line of the parasite stably in the laboratory. Here, we summarize and compare the available methodologies and conditions for the in vitro cultivation of P. vivax.     

Species-specific, postentry barriers to primate immunodeficiency virus infection

By using replication-defective vectors derived from human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus (SIV(mac)), and murine leukemia virus (MuLV), all of which were pseudotyped with the vesicular stomatitis virus (VSV) G glycoprotein, the efficiency of postentry, early infection events was examined in target cells of several mammalian species. Titers of HIV-1 vectors were significantly lower than those of SIV(mac) and MuLV vectors in most cell lines and primary cells from Old World monkeys. By contrast, most New World monkey cells exhibited much lower titers for the SIV(mac) vector compared with those of the HIV-1 vector. Prosimian cells were resistant to both HIV-1 and SIV(mac) vectors, although the MuLV vector was able to infect these cells. Cells from other mammalian species were roughly equivalent in susceptibility to the three vectors, with the exception of rabbit cells, which were specifically resistant to the HIV-1 vector. The level of HIV-1 vector expression was very low in transduced cells of rodent, rabbit, cow, and pig origin. Early postentry restriction of primate immunodeficiency virus infection exhibits patterns largely coincident with species borders and applies to diverse cell types within an individual host, suggesting the involvement of species-specific, widely expressed cellular factors.     

Cloning, overexpression, purification and characterization of Plasmodium knowlesi lactate dehydrogenase

Plasmodial lactate dehydrogenase, key enzyme of anaerobic glycolysis, has been shown to be a potential immunodiagnostic marker as well as a novel target for chemotherapy. We have cloned, overexpressed and immunochemically characterized the recombinant lactate dehydrogenase of Plasmodium knowlesi, the fifth human malaria parasite. The P. knowlesi lactate dehydrogenase (PkLDH) gene was PCR amplified and 0.9 kb PCR product was cloned into pGEM-T Easy vector. Sequencing and BLAST analysis revealed open reading frame of 316 amino acids of PkLDH showing 96.8% homology with Plasmodium vivax LDH and around 90% with Plasmodium falciparum, Plasmodium malariae and Plasmodium ovale LDHs. The PkLDH gene was subcloned into pGEX-6P1 expression vector and the SDS-PAGE analysis revealed that about 70% of fusion protein was present in the soluble fraction. The fusion protein was cleaved with PreScission protease and recombinant PkLDH (34 kDa) was affinity purified to homogeneity. The purified PkLDH exhibited high reactivity with polyclonal and monoclonal antibodies against plasmodial LDH. The polyclonal antibody produced against purified recombinant PkLDH in rabbits showed high ELISA reactivity with both native and recombinant PkLDH and could detect parasite LDH in malaria infected blood samples by sandwich ELISA. The purified recombinant PkLDH can be used to produce P. knowlesi specific monoclonal antibodies for specific diagnosis of P. knowlesi infection in humans.     

Gametocytemia in Plasmodium vivax and Plasmodium falciparum infections

Two expert research microscopists, each blinded to the other's reports, diagnosed single-species malaria infections in 2,141 adults presenting at outpatient malaria clinics in Tak Province, Thailand, and Iquitos, Peru, in May-August 1998, May-July 1999, and May-June 2001. Plasmodium vivax patients with gametocytemia had higher fever and higher parasitemia than those without gametocytemia; temperature correlated with parasitemia in the patients with gametocytemia. Plasmodium falciparum patients with gametocytemia had lower fever than those without gametocytemia, but similar parasitemia; temperature correlated with parasitemia in the patients without gametocytemia. Hematologic data in Thailand in 2001 showed lower platelet counts in P. vivax patients with gametocytemia than in the P. vivax patients without gametocytemia, whereas P. falciparum patients with gametocytemia had similar platelet counts but lower red blood cell counts, hemoglobin levels, hematocrit levels, and higher lymphocyte counts than patients without gametocytemia.     

Platform for Plasmodium vivax vaccine discovery and development

Plasmodium vivax is the most prevalent malaria parasite on the American continent. It generates a global burden of 80-100 million cases annually and represents a tremendous public health problem, particularly in the American and Asian continents. A malaria vaccine would be considered the most cost-effective measure against this vector-borne disease and it would contribute to a reduction in malaria cases and to eventual eradication. Although significant progress has been achieved in the search for Plasmodium falciparum antigens that could be used in a vaccine, limited progress has been made in the search for P. vivax components that might be eligible for vaccine development. This is primarily due to the lack of in vitro cultures to serve as an antigen source and to inadequate funding. While the most advanced P. falciparum vaccine candidate is currently being tested in Phase III trials in Africa, the most advanced P. vivax candidates have only advanced to Phase I trials. Herein, we describe the overall strategy and progress in P. vivax vaccine research, from antigen discovery to preclinical and clinical development and we discuss the regional potential of Latin America to develop a comprehensive platform for vaccine development.     

Protective antigens of bloodstage Plasmodium knowlesi parasites

The simian malaria Plasmodium knowlesi provides many favourable features as an experimental model; it can be grown in vivo or in vitro. Parasites of defined variant specificity and stage of development are readily obtained and both the natural host and a highly susceptible host are available for experimental infection and vaccination trials. Proteins synthesized by erythrocytic P. knowlesi parasites are characteristic of the developmental stage, as are the alterations that the parasite induces in the red cell surface. Erythrocytic merozoites are anatomically and biochemically complex, their surface alone is covered by at least eight distinct polypeptides. Immune serum from merozoite-immunized rhesus recognizes many parasite components, especially those synthesized by schizonts. All of the merozoite surface components and some of the schizont-infected red cell surface antigens are recognized by such immune sera. Rhesus monkeys rendered immune by repeated infection may by contrast recognize comparatively few antigens; a positive correlation was established for these 'naturally' immunized monkeys between protection and antibody directed against a 74 000 molecular mass antigen. Immunization with this purified antigen confers partial protection. Other putative protective antigens have been identified by monoclonal antibodies that inhibit merozoite invasion of red cells in vitro. The antigens recognized by inhibitory monoclonal antibodies are synthesized exclusively by schizonts and are processed, at the time of schizont rupture and merozoite release, to smaller molecules that are present on the merozoite surface. The multiplicity of protective antigens is clearly demonstrated by the fact that seven distinct merozoite surface antigens are recognized by three different inhibitory monoclonals. None of the protective antigens identified are variant or strain specific.     

Neglect of Plasmodium vivax malaria

Plasmodium vivax infects 130-435 million of the 2.6 billion people living at risk of infection. Recent studies suggest that vivax malaria can become lethal in a similar way to severe falciparum malaria. First-line therapies remain unchanged after 50 years. Despite evidence of failing chloroquine efficacy, little work has assessed the problem or explored alternative therapies. Primaquine treatment, the only therapeutic option against relapse, might also be failing. No licensed primary chemoprophylactic agent protects travelers from relapse. Misdiagnosis of species now affects clinical decisions resulting in inadequate therapy for P. falciparum and P. vivax. All of these factors demonstrate the lack of research on P. vivax.     

Targeting the Plasmodium vivax Duffy-binding protein

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

Identifying and characterising the Plasmodium falciparum RhopH3 Plasmodium vivax homologue

Four Plasmodium species cause malaria in humans, Plasmodium falciparum being the most widely studied to date. All Plasmodium species have paired club-shaped organelles towards their apical extreme named rhoptries that contain many lipids and proteins which are released during target cell invasion. P. falciparum RhopH3 is a rhoptry protein triggering important immune responses in patients from endemic regions. It has also been shown that anti-RhopH3 antibodies inhibit in vitro invasion of erythrocytes. Recent immunisation studies in mice with the Plasmodium yoelii and Plasmodium berghei RhopH3 P. falciparum homologue proteins found that they are able to induce protection in murine models. This study described identifying and characterising RhopH3 protein in Plasmodium vivax; it is encoded by a seven exon gene and expressed during the parasite's asexual stage. PvRhopH3 has similar processing to its homologue in P. falciparum and presents a cellular immunolocalisation pattern characteristic of rhoptry proteins.     

Fine structure of the erythrocytic stages of Plasmodium knowlesi

Summary The fine structure of erythrocytic stages of Plasmodium knowlesi was compared with that of the same parasite isolated from its host cell by a saponin technique. Rhesus monkeys experimentally infected with Plasmodium knowlesi were the source of parasitized red cells. The erythrocytic stages of this Plasmodium showed all the organelles described in other mammalian forms; the nucleus lacked a typical nucleolus but contained a cluster of granules. P. knowlesi did not have protozoan-type mitochondria as do the avian and reptilian forms, but had double-membrane-bounded bodies as observed in other mammalian malarial parasites.The isolation procedure caused a slight swelling of the parasite, but in general, the structure and structural relationships of the parasite were preserved. However, the isolation technique gave a new insight into the connection of the host cell cytoplasm with the large, so-called food vacuoles of the parasite. The parasite freed from its host cell showed clear spaces where the large vacuoles had been. The content of these vacuoles had been removed together with the red cell cytoplasm. As the nature of the isolation procedure precluded any disruption of the parasite itself, these findings support our view that the vacuoles are not true food vacuoles. If these were true food vacuoles, they would be completely enclosed by a parasite membrane within the parasite cytoplasm. However, we have demonstrated that they represent extensions of host cell cytoplasm in direct communication with the rest of the red cell. The outer membrane surrounding the intra-erythrocytic parasites disappeared after isolation of the parasite from the host cell. This strongly suggested that the outer membrane is of host cell origin. The budding process of the merozoites from a schizont was also described and discussed.This paper is contribution No. 558 from the Army Research Program on Malaria and was supported in part by Research Grant AI 08970-01 from the United States Public Health Service.