The Baculovirus-Expressed Binding Region of Plasmodium falciparum EBA-140 Ligand and Its Glycophorin C Binding Specificity

The erythrocyte binding ligand 140 (EBA-140) is a member of the Plasmodium falciparum DBL family of erythrocyte binding proteins, which are considered as prospective candidates for malaria vaccine development. The EBA-140 ligand is a paralogue of the well-characterized P. falciparum EBA-175 protein. They share homology of domain structure, including Region II, which consists of two homologous F1 and F2 domains and is responsible for ligand-erythrocyte receptor interaction during invasion. In this report we describe, for the first time, the glycophorin C specificity of the recombinant, baculovirus-expressed binding region (Region II) of P. falciparum EBA-140 ligand. It was found that the recombinant EBA-140 Region II binds to the endogenous and recombinant glycophorin C, but does not bind to Gerbich-type glycophorin C, neither normal nor recombinant, which lacks amino acid residues 36–63 of its polypeptide chain. Our results emphasize the crucial role of this glycophorin C region in EBA-140 ligand binding. Moreover, the EBA-140 Region II did not bind either to glycophorin D, the truncated form of glycophorin C lacking the N-glycan or to desialylated GPC. These results draw attention to the role of glycophorin C glycans in EBA-140 binding. The full identification of the EBA-140 binding site on glycophorin C molecule, consisting most likely of its glycans and peptide backbone, may help to design therapeutics or vaccines that target the erythrocyte binding merozoite ligands.     

Delineation of stage specific expression of Plasmodium falciparum EBA-175 by biologically functional region II monoclonal antibodies

Background

The malaria parasite Plasmodium falciparum EBA-175 binds its receptor sialic acids on glycophorin A when invading erythrocytes. The receptor-binding region (RII) contains two cysteine-rich domains with similar cysteine motifs (F1 and F2). Functional relationships between F1 and F2 domains and characterization of EBA-175 were studied using specific monoclonal antibodies (mAbs) against these domains.

Methods and Findings

Five mAbs specific for F1 or F2 were generated. Three mAbs specific for F2 potently blocked binding of EBA-175 to erythrocytes, and merozoite invasion of erythrocytes (IC₅₀ 10 to 100 µg/ml IgG in growth inhibition assays). A mAb specific for F1 blocked EBA-175 binding and merozoite invasion less effectively. The difference observed between the IC₅₀ of F1 and F2 mAbs was not due to differing association and disassociation rates as determined by surface plasmon resonance. Four of the mAbs recognized conformation-dependent epitopes within F1 or F2. Used in combination, F1 and F2 mAbs blocked the binding of native EBA-175 to erythrocytes and inhibited parasite invasion synergistically in vitro. MAb R217, the most potent, did not recognize sporozoites, 3-day hepatocyte stage parasites, nor rings, trophozoites, gametocytes, retorts, ookinetes, and oocysts but recognized 6-day hepatocyte stage parasites, and schizonts. Even though efficient at blocking binding to erythrocytes and inhibiting invasion into erythrocytes, MAb R217 did not inhibit sporozoite invasion and development in hepatocytes in vitro.

Conclusions

The role of the F1 and F2 domains in erythrocyte invasion and binding was elucidated with mAbs. These mAbs interfere with native EBA-175 binding to erythrocyte in a synergistic fashion. The stage specific expression of EBA-175 showed that the primary focus of activity was the merozoite stage. A recombinant RII protein vaccine consisting of both F1 and F2 domains that could induce synergistic activity should be optimal for induction of antibody responses that interfere with merozoite invasion of erythrocytes.     

The Plasmodium falciparum Erythrocyte Invasion Ligand Pfrh4 as a Target of Functional and Protective Human Antibodies against Malaria

Background

Acquired antibodies are important in human immunity to malaria, but key targets remain largely unknown. Plasmodium falciparum reticulocyte-binding-homologue-4 (PfRh4) is important for invasion of human erythrocytes and may therefore be a target of protective immunity.

Methods

IgG and IgG subclass-specific responses against different regions of PfRh4 were determined in a longitudinal cohort of 206 children in Papua New Guinea (PNG). Human PfRh4 antibodies were tested for functional invasion-inhibitory activity, and expression of PfRh4 by P. falciparum isolates and sequence polymorphisms were determined.

Results

Antibodies to PfRh4 were acquired by children exposed to P. falciparum malaria, were predominantly comprised of IgG1 and IgG3 subclasses, and were associated with increasing age and active parasitemia. High levels of antibodies, particularly IgG3, were strongly predictive of protection against clinical malaria and high-density parasitemia. Human affinity-purified antibodies to the binding region of PfRh4 effectively inhibited erythrocyte invasion by P. falciparum merozoites and antibody levels in protected children were at functionally-active concentrations. Although expression of PfRh4 can vary, PfRh4 protein was expressed by most isolates derived from the cohort and showed limited sequence polymorphism.

Conclusions

Evidence suggests that PfRh4 is a target of antibodies that contribute to protective immunity to malaria by inhibiting erythrocyte invasion and preventing high density parasitemia. These findings advance our understanding of the targets and mechanisms of human immunity and evaluating the potential of PfRh4 as a component of candidate malaria vaccines.     

Crystal and Solution Structures of Plasmodium falciparum Erythrocyte-binding Antigen 140 Reveal Determinants of Receptor Specificity during Erythrocyte Invasion

Erythrocyte-binding antigen 140 (PfEBA-140) is a critical Plasmodium falciparum erythrocyte invasion ligand that engages glycophorin C on host erythrocytes during malaria infection. The minimal receptor-binding region of PfEBA-140 contains two conserved Duffy binding-like (DBL) domains, a fold unique to Plasmodium species. Here, we present the crystal structure of the receptor-binding region of PfEBA-140 at 2.4 Å resolution. The two-domain binding region is present as a monomer in the asymmetric unit, and the structure reveals novel features in PfEBA-140 that are likely determinants of receptor specificity. Analysis by small-angle x-ray scattering demonstrated that the minimal binding region is monomeric in solution, consistent with the crystal structure. Erythrocyte binding assays showed that the full-length binding region containing the tandem DBL domains is required for erythrocyte engagement, suggesting that both domains contain critical receptor contact sites. The electrostatic surface of PfEBA-140 elucidates a basic patch that constitutes a putative high-affinity binding interface spanning both DBL domains. Mutation of residues within this interface results in severely diminished erythrocyte binding. This study provides insight into the structural basis and mechanism of PfEBA-140 receptor engagement and forms a basis for future studies of this critical interaction. In addition, the solution and crystal structures allow the first identification of likely determinants of erythrocyte receptor specificity for P. falciparum invasion ligands. A complete understanding of the PfEBA-140 erythrocyte invasion pathway will aid in the design of invasion inhibitory therapeutics and vaccines.     

The cytoplasmic domain of the Plasmodium falciparum ligand EBA-175 is essential for invasion but not protein trafficking

The invasion of host cells by the malaria parasite Plasmodium falciparum requires specific protein-protein interactions between parasite and host receptors and an intracellular translocation machinery to power the process. The transmembrane erythrocyte binding protein-175 (EBA-175) and thrombospondin-related anonymous protein (TRAP) play central roles in this process. EBA-175 binds to glycophorin A on human erythrocytes during the invasion process, linking the parasite to the surface of the host cell. In this report, we show that the cytoplasmic domain of EBA-175 encodes crucial information for its role in merozoite invasion, and that trafficking of this protein is independent of this domain. Further, we show that the cytoplasmic domain of TRAP, a protein that is not expressed in merozoites but is essential for invasion of liver cells by the sporozoite stage, can substitute for the cytoplasmic domain of EBA-175. These results show that the parasite uses the same components of its cellular machinery for invasion regardless of the host cell type and invasive form.     

Enhancing Blockade of Plasmodium falciparum Erythrocyte Invasion: Assessing Combinations of Antibodies against PfRH5 and Other Merozoite Antigens

No vaccine has yet proven effective against the blood-stages of Plasmodium falciparum, which cause the symptoms and severe manifestations of malaria. We recently found that PfRH5, a P. falciparum-specific protein expressed in merozoites, is efficiently targeted by broadly-neutralizing, vaccine-induced antibodies. Here we show that antibodies against PfRH5 efficiently inhibit the in vitro growth of short-term-adapted parasite isolates from Cambodia, and that the EC₅₀ values of antigen-specific antibodies against PfRH5 are lower than those against PfAMA1. Since antibody responses elicited by multiple antigens are speculated to improve the efficacy of blood-stage vaccines, we conducted detailed assessments of parasite growth inhibition by antibodies against PfRH5 in combination with antibodies against seven other merozoite antigens. We found that antibodies against PfRH5 act synergistically with antibodies against certain other merozoite antigens, most notably with antibodies against other erythrocyte-binding antigens such as PfRH4, to inhibit the growth of a homologous P. falciparum clone. A combination of antibodies against PfRH4 and basigin, the erythrocyte receptor for PfRH5, also potently inhibited parasite growth. This methodology provides the first quantitative evidence that polyclonal vaccine-induced antibodies can act synergistically against P. falciparum antigens and should help to guide the rational development of future multi-antigen vaccines.     

Analysis of a Plasmodium falciparum EBA-175 peptide with high binding capacity to erythrocytes and their analogues using 1H NMR

A 175-erythrocyte-binding protein (EBA-175) conserved high-activity binding peptide (HABP), called 1783 (nonimmunogenic, nonprotective against Plasmodium falciparum malaria), was analyzed for antigenic and protective activity in Aotus monkeys, together with several of its analogues. 1H NMR studies of peptides 17912, 14016, and 22814 allowed their structure to be related to their biological function. These peptides showed helical regions having differences in their position and length. Nonimmunogenic, nonprotective peptides 1783 and 17912 showed an extensive helical region, while the 22814 immunogenic protective peptide's alpha-helix was found in the N-terminal region. This suggests that the more flexible C-terminal region will allow better interaction between these peptides and immune system molecules as well as relating these peptides' three-dimensional structure to their immunogenicity and protective activity, thus leading to a more rational development of the new malaria multicomponent vaccine.     

Induction of biologically active antibodies in mice, rabbits, and monkeys by Plasmodium falciparum EBA-175 region II DNA vaccine

BACKGROUND: Plasmodium falciparum merozoites bind to and invade human erythrocytes via specific erythrocyte receptors. This establishes the erythrocytic stage of the parasite life cycle that causes clinical disease resulting in 2-3 million deaths per year. We tested the hypothesis that a Plasmodium falciparum ligand, EBA-175 region II (RII), which binds its erythrocyte receptor glycophorin A during invasion, can be used as an immunogen to induce antibodies that block the binding of RII to erythrocytes and thereby inhibit parasite invasion of erythrocytes. Accordingly, we immunized mice, rabbits, and monkeys with DNA plasmids that encoded the 616 amino acid RII. MATERIALS AND METHODS: DNA vaccine plasmids that targeted the secretion of recombinant RII protein with and without the universal T-cell helper epitopes P2P30 were used to immunize mice, rabbits, and Aotus monkeys. RII specific antibodies were assessed by IFA, ELISA, blocking of native [35S] labeled EBA-175 binding to human erythrocytes, and growth inhibition assays, all in vitro. RESULTS: The RII DNA plasmids were highly immunogenic as measured by ELISA and IFA. The anti-RII antibodies blocked the binding of native EBA-175 to erythrocytes, and rosetting of erythrocytes on COS-7 cells expressing RII. Most important, murine and rabbit anti-RII antibodies inhibited the invasion of merozoites into erythrocytes. We immunized nonhuman primates and showed that the RII-DNA plasmids were immunogenic and well tolerated in these monkeys. Monkeys were challenged with parasitized erythrocytes; one of three monkeys that received RII DNA plasmid was protected from fulminant disease. After challenge with live parasites, anti-RII antibody titers were boosted in the immunized monkeys. CONCLUSIONS: By proving the hypothesis that anti-RII antibodies can block merozoite invasion of erythrocytes, these studies pave the way for the clinical evaluation of EBA-175 as a receptor-blockade vaccine.     

Immunogenicity and protectivity of Plasmodium falciparum EBA-175 peptide and its analog is associated with alpha-helical region shortening and displacement

EBA-175 protein is used as a ligand in the binding of P. falciparum to red blood cells (RBCs). Evidence shows that the conserved peptide 1779 from this protein (with high red blood cell binding ability and known critical erythrocyte binding residues) plays an important role in the invasion process. This peptide is neither immunogenic nor protective; analogs having critical residues replaced by amino acids with similar volume or mass but different polarity were synthesized and inoculated into Aotus monkeys, and elicited different immunogenic and protective responses. Nuclear Magnetic Resonance (1H-NMR) studies revealed that peptide analog 21696 (non-immunogenic and non-protective) presents a large helical fragment, that the peptide 14012 (immunogenic and non-protective) helical fragment is smaller, while the peptide 22812 (immunogenic and protective) alpha-helix is shorter in a different region and possesses greater flexibility at its N-terminus. The presence of methionine residues could affect the structural stability of peptide 22812 and ultimately its immunological response. Our results suggest a new strategy for designing a new malaria multi-component subunit-based vaccine.     

The Binding of Bispecific Monoclonal Antibodies to the Solid Phase-Adsorbed Antigens

The ability of bispecific antibodies (Babs) formed by fusion of hybridomas and parent monoclonal antibodies (Mabs) to interact with the solid phase-adsorbed antigens was studied. Mabs specific to the three different antigens [horseradish peroxidase (HRP), human IgG (hIgG), and human myoglobin (Mb)] as well as Babs with the double specificity [antimyoglobin/antiperoxidase (anti-Mb/HRP) and anti-human IgG/antiperoxidase (anti-hIgG/HRP)] were used. It was shown by radioimmunological and immunoenzyme assays that parent Mabs bind to solid phase-adsorbed antigens considerably more effectively than Babs. The observed equilibrium binding constant (K _a) of antiperoxidase parental Mabs to immobilized HRP is 21 and 38 times higher than K _afor Babs binding sites (anti-Mb/HRP and anti-hIgG/HRP, respectively) to peroxidase. It was calculated that about 90–95% of all bound parental antiperoxidase Mabs were associated with immobilized HRP bivalently, and only about 5–10% were bound monovalently. On the contrary, parental Mabs against hIgG bind to the sorbed antigen essentially only monovalently. It was also shown that the avidity of anti-Mb/HRP Babs significantly increased when two antigens, Mb and HRP, were simultaneously adsorbed on the solid phase. These data imply that Babs bearing an enzyme-binding site (for example, binding to HRP) cannot be more effective than standard conjugates (e.g., enzyme-conjugated antibodies) in heterogeneous noncompetitive immunoassays.     

Using Mutagenesis and Structural Biology to Map the Binding Site for the Plasmodium falciparum Merozoite Protein PfRh4 on the Human Immune Adherence Receptor

To survive and replicate within the human host, malaria parasites must invade erythrocytes. Invasion can be mediated by the P. falciparum reticulocyte-binding homologue protein 4 (PfRh4) on the merozoite surface interacting with complement receptor type 1 (CR1, CD35) on the erythrocyte membrane. The PfRh4 attachment site lies within the three N-terminal complement control protein modules (CCPs 1–3) of CR1, which intriguingly also accommodate binding and regulatory sites for the key complement activation-specific proteolytic products, C3b and C4b. One of these regulatory activities is decay-accelerating activity. Although PfRh4 does not impact C3b/C4b binding, it does inhibit this convertase disassociating capability. Here, we have employed ELISA, co-immunoprecipitation, and surface plasmon resonance to demonstrate that CCP 1 contains all the critical residues for PfRh4 interaction. We fine mapped by homologous substitution mutagenesis the PfRh4-binding site on CCP 1 and visualized it with a solution structure of CCPs 1–3 derived by NMR and small angle x-ray scattering. We cross-validated these results by creating an artificial PfRh4-binding site through substitution of putative PfRh4-interacting residues from CCP 1 into their homologous positions within CCP 8; strikingly, this engineered binding site had an ∼30-fold higher affinity for PfRh4 than the native one in CCP 1. These experiments define a candidate site on CR1 by which P. falciparum merozoites gain access to human erythrocytes in a non-sialic acid-dependent pathway of merozoite invasion.     

Anti-Human IgE Monoclonal Antibodies Recognizing Epitopes Related to the Binding Sites of High and Low Affinity IgE Receptors

Anti-human IgE monoclonal antibodies (mAbs) were produced and eight clones recognizing epitopes on native IgE were selected. Epitopes were mapped by a competitive inhibition enzyme-linked immunosorbent assay, Western blotting and a multi-pin peptide technology. Four sites (one each in the Cε1, Cε2, Cε2/Cε3 junction and Cε3) were recognized by the mAbs. The relationship between the four epitopes and the binding sites of high and low affinity IgE receptors (FcεRI and FcεRII, respectively) was studied using a monovalent Fab fragment of each mAb as a binding inhibitor. The IgE-FcεRII binding was clearly inhibited by the mAb recognizing the Cε2/Cε3 junction, suggesting that FcεRII binds to a rather limited area around the Cε2/Cε3 junction. The IgE-FcεRI binding, on the other hand, was scarcely inhibited by any single mAb. However, the binding was inhibited when the epitope in Cε2 was blocked simultaneously with that at the Cε2/Cε3 junction or with that in Cε3, indicating that these three distinct epitopes are related to the FcεRI binding sites. When these three epitopes were shown in the stereograph of human IgE, the FcεRI binding area was spread largely on the groove side between Cε2 and Cε3 domains. These results suggest that FcεRI acquires the high affinity through multiple bindings.     

Preparation and Characterization of Monoclonal Antibodies to Serotonin Binding Protein

Serotonin binding protein (SBP) is a constituent of the synaptic vesicles of serotonergic neurons. Two types of SBP, with molecular masses of 45 kDa and 56 kDa, have been purified. To determine whether there are shared epitopes between the two forms of SBP, we raised and tested for cross-reactivity monoclonal antibodies (MAbs) against each form of SBP. We obtained 12 MAbs, all of which recognize both forms of SBP. Hybridoma clones were produced by fusing P3 X 63Ag8.653 mouse myeloma cells with spleen cells from a mouse that had been immunized with 45-kDa or 56-kDa SBP. Culture supernatants were screened for the presence of anti-SBP antibodies. MAb isotypes were determined by immunodiffusion, using immunoglobulin type-specific antisera. Each antibody to SBP consisted of only a single subclass of immunoglobulin (IgM). We obtained 12 MAbs, each of which interacted with both forms of SBP, as judged by enzyme-linked immunosorbent assay and immunoblot analysis. Ascites fluid to one clone (44-10) was obtained and affinity-purified. In the presence of goat anti-mouse IgM, the partially purified 44-10 antibodies quantitatively immunoprecipitated SBP from crude brain extracts. Immunoblotting revealed two major bands corresponding to 45 kDa and 56 kDa and a minor band corresponding to 68 kDa. MAb 44-10 blocked the binding of [3H]serotonin ([3H]5-HT) to 45-kDa and 56-kDa SBP in a concentration-dependent manner. The 68-kDa protein was found to bind [3H]5-HT. Sites reacting with MAB 44-10 were located immunocytochemically in sections of rat brain.(ABSTRACT TRUNCATED AT 250 WORDS)     

甲型H1N1流感病毒HA蛋白抗原表位及受体结合位点突变特性的对比研究

人群中流行的H1N1病毒按其来源可分为两类:人感染的猪H1N1病毒与人类季节性H1N1流感病毒。这两类病毒在流行频率、易感性和致病性等方面存在明显差异。文章收集了1918～2009年间17株人感染的猪甲型H1N1毒株以及21株季节性H1N1毒株,通过序列比对、氨基酸残基保守性分析及3D结构对比等生物信息学方法,揭示造成这两类病毒流行病学和感染性差异的机制。研究发现这两类病毒HA蛋白的进化路径并不相同,且两者具有不同的突变特征,人感染的猪H1N1病毒中,Ca1、Ca2、Sa和Sb四个位点均较为保守,仅Cb位点的突变较快;季节性H1N1病毒仅有Ca1位点较为保守,其他四个抗原性位点均具有较快的突变速率,且较多的突变为新类型的氨基酸。另外,对受体结合位点的研究也显示,这两类病毒的该区域存在5个氨基酸水平的差异(ALA138SER、GLN192LYS、GLN196HIS、ALA198GLU和ALA227GLU),这些位点的差异使得人感染的猪H1N1流感病毒比人类季节性H1N1病毒的易感性更强。这些研究结果可为阐明两类H1N1流感病毒感染性及致病性差异提供更多的信息,并有助于进一步认识H1N1流感病毒的进化机制。     

Characteristics of Monoclonal Antibodies to Lucerne Transient Streak Sobemovirus and Their Use in Epitope Localization

Murine monoclonal antibodies (mAbs) to lucerne transient streak sobemovirus (LTSV) were used as probes for examining the virion capsid organization. A panel of nine mAbs from approximately 100 hybridomas were chosen for this study. All of these mAbs interacted effectively with sites on intact capsid and isolated coat protein sub-units (i.e. metatopes) in ELISA and Western blots. Only one of these mAbs reacted with virions in gel diffusion test producing a visible precipitin band. Competitive binding assays showed that these mAbs recognized the same or very similar metatopes. None of the mAbs neutralized LTSV virion infectivity but rabbit polyclonal antibodies drastically reduced infectivity. Treatment of virions with EDTA (swollen LTSV). protein crosslinking reagents or sodium dodecyl sulphate caused no detectable alterations in their reactivities with these mAbs, The binding sites for monoclonal and polyclonal antibodies were located on denatured 5.5 kDa fragments resulting from partial trypsinization of LTSV coat protein and 8 kDa fragments formed with chymopapain proteolysis. These results indicate that these LTSV epitopes are of linear (or sequential) configuration and are located on the exposed, structurally stable domain of the virion capsid.     

Characterization of Plasmodium falciparum Calcium-dependent Protein Kinase 1 (PfCDPK1) and Its Role in Microneme Secretion during Erythrocyte Invasion

Calcium-dependent protein kinases (CDPKs) play important roles in the life cycle of Plasmodium falciparum and other apicomplexan parasites. CDPKs commonly have an N-terminal kinase domain (KD) and a C-terminal calmodulin-like domain (CamLD) with calcium-binding EF hands. The KD and CamLD are separated by a junction domain (JD). Previous studies on Plasmodium and Toxoplasma CDPKs suggest a role for the JD and CamLD in the regulation of kinase activity. Here, we provide direct evidence for the binding of the CamLD with the P3 region (Leu³⁵⁶ to Thr³⁷⁰) of the JD in the presence of calcium (Ca²⁺). Moreover, site-directed mutagenesis of conserved hydrophobic residues in the JD (F363A/I364A, L356A, and F350A) abrogates functional activity of PfCDPK1, demonstrating the importance of these residues in PfCDPK1 function. Modeling studies suggest that these residues play a role in interaction of the CamLD with the JD. The P3 peptide, which specifically inhibits the functional activity of PfCDPK1, blocks microneme discharge and erythrocyte invasion by P. falciparum merozoites. Purfalcamine, a previously identified specific inhibitor of PfCDPK1, also inhibits microneme discharge and erythrocyte invasion, confirming a role for PfCDPK1 in this process. These studies validate PfCDPK1 as a target for drug development and demonstrate that interfering with its mechanistic regulation may provide a novel approach to design-specific PfCDPK1 inhibitors that limit blood stage parasite growth and clear malaria parasite infections.     

MiyabenolC和KobophenolA与雌激素受体的结合位点

iyabenolC (MiyC)和kobophenolA (KobA)是两种新型的植物雌激素。为了探讨MiyC和KobA与雌激素受体 (ER)的结合部位 ,运用计算机辅助分子模拟构建它们与ER结合的空间模型 ,找出结合位点 ,设计ER的两个突变体M1ER(ERM517AG52 1D)和M2ER(ERE353GR394 G) ;运用PCR技术将ER与MiyC或KobA的结合位点进行突变 ;运用报告基因检测实验 ,检测MiyC和KobA对突变的ER是否具有激活功能。结果显示MiyC激活M1ER使之促下游基因转录的作用下降 ,KobA对M1ER无激活作用 ;MiyC和KobA对M2ER无激活作用。以上结果显示MiyC和KobA与ER的结合位点可能为ER的Glu353 、Arg394 、Met517和Gly52 1。     

The structure of the Plasmodium falciparum EBA175 ligand domain and the molecular basis of host specificity

Erythrocyte-binding antigen 175 (EBA175) is one of the best-characterized Plasmodium falciparum merozoite ligands; the recently solved crystal structure of EBA175 reveals that terminal sialic acids on the erythrocyte glycoprotein glycophorin A are a crucial factor for erythrocyte recognition by EBA175 because they lock into pockets on its surface. Comparison with Plasmodium reichenowi EBA175 indicates that these interactions have a pivotal role in the host-specific adaptations of parasite ligands.