RESUMO
The human malaria parasite, Plasmodium falciparum, is able to evade spleen-mediated clearing from blood stream by sequestering in peripheral organs. This is due to the adhesive properties conferred by the P. falciparum Erythrocyte Membrane Protein 1 (PfEMP1) family exported by the parasite to the surface of infected erythrocytes. Expression of the VAR2CSA variant of PfEMP1 leads to pregnancy-associated malaria, which occurs when infected erythrocytes massively sequester in the placenta by binding to low-sulfated Chondroitin Sulfate A (CSA) present in the intervillous spaces. VAR2CSA is a 350 kDa protein that carries six Duffy-Binding Like (DBL) domains, one Cysteine-rich Inter-Domain Regions (CIDR) and several inter-domain regions. In the present paper, we report for the first time the crystal structure at 2.9 Šof a VAR2CSA double domain, DBL3X-DBL4ε, from the FCR3 strain. DBL3X and DBL4ε share a large contact interface formed by residues that are invariant or highly conserved in VAR2CSA variants, which suggests that these two central DBL domains (DBL3X-DBL4ε) contribute significantly to the structuring of the functional VAR2CSA extracellular region. We have also examined the antigenicity of peptides corresponding to exposed loop regions of the DBL4ε structure.
Assuntos
Antígenos de Protozoários/imunologia , Vacinas Antimaláricas/imunologia , Malária Falciparum/imunologia , Placenta/imunologia , Plasmodium falciparum/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antiprotozoários/sangue , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/química , Antígenos de Protozoários/genética , Sítios de Ligação/genética , Sítios de Ligação/imunologia , Sulfatos de Condroitina/imunologia , Sulfatos de Condroitina/metabolismo , Cristalografia por Raios X , Eritrócitos/imunologia , Eritrócitos/parasitologia , Feminino , Interações Hospedeiro-Parasita/imunologia , Humanos , Soros Imunes/imunologia , Vacinas Antimaláricas/administração & dosagem , Malária Falciparum/parasitologia , Malária Falciparum/prevenção & controle , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Placenta/metabolismo , Placenta/parasitologia , Plasmodium falciparum/metabolismo , Plasmodium falciparum/fisiologia , Gravidez , Ligação Proteica/imunologia , Estrutura Terciária de Proteína , Coelhos , Homologia de Sequência de AminoácidosRESUMO
Adhesion of Plasmodium falciparum-infected red blood cells (iRBC) to human erythrocytes (i.e. rosetting) is associated with severe malaria. Rosetting results from interactions between a subset of variant PfEMP1 (Plasmodium falciparum erythrocyte membrane protein 1) adhesins and specific erythrocyte receptors. Interfering with such interactions is considered a promising intervention against severe malaria. To evaluate the feasibility of a vaccine strategy targetting rosetting, we have used here the Palo Alto 89F5 VarO rosetting model. PfEMP1-VarO consists of five Duffy-Binding Like domains (DBL1-5) and one Cysteine-rich Interdomain Region (CIDR1). The binding domain has been mapped to DBL1 and the ABO blood group was identified as the erythrocyte receptor. Here, we study the immunogenicity of all six recombinant PfEMP1-VarO domains and the DBL1- CIDR1 Head domain in BALB/c and outbred OF1 mice. Five readouts of antibody responses are explored: ELISA titres on the recombinant antigen, VarO-iRBC immunoblot reactivity, VarO-iRBC surface-reactivity, capacity to disrupt VarO rosettes and the capacity to prevent VarO rosette formation. For three domains, we explore influence of the expression system on antigenicity and immunogenicity. We show that correctly folded PfEMP1 domains elicit high antibody titres and induce a homogeneous response in outbred and BALB/c mice after three injections. High levels of rosette-disrupting and rosette-preventing antibodies are induced by DBL1 and the Head domain. Reduced-alkylated or denatured proteins fail to induce surface-reacting and rosette-disrupting antibodies, indicating that surface epitopes are conformational. We also report limited cross-reactivity between some PfEMP1 VarO domains. These results highlight the high immunogenicity of the individual domains in outbred animals and provide a strong basis for a rational vaccination strategy targeting rosetting.
Assuntos
Adesinas Bacterianas/imunologia , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Adesinas Bacterianas/química , Adesinas Bacterianas/genética , Animais , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/química , Antígenos de Protozoários/genética , Reações Cruzadas , Epitopos/química , Epitopos/genética , Eritrócitos/parasitologia , Feminino , Humanos , Vacinas Antimaláricas/química , Vacinas Antimaláricas/genética , Vacinas Antimaláricas/imunologia , Malária Falciparum/imunologia , Malária Falciparum/parasitologia , Malária Falciparum/prevenção & controle , Camundongos , Camundongos Endogâmicos BALB C , Plasmodium falciparum/genética , Plasmodium falciparum/patogenicidade , Estrutura Terciária de Proteína , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Formação de RosetaRESUMO
Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), a family of adhesins of the falciparum species of the malaria parasite, is exposed on the surface of the infected erythrocyte. In general, only one PfEMP1 variant is expressed at a time but switching between variants occurs, changing both host-cell receptor specificity and serotype. The PfEMP1 variant VAR2CSA causes sequestration of infected erythrocytes in the intervillous spaces of the placenta via the glycosaminoglycan chondroitin sulfate A. This leads to pregnancy-associated malaria, which has severe consequences for the fetus and mother. The extracellular region of VAR2CSA comprises six DBL (Duffy-binding-like) domains and a single CIDR (cysteine-rich inter-domain region) domain. The C-terminal domain DBL6ε, the most polymorphic domain of VAR2CSA, has seven regions of high variability termed variable blocks (VBs). Here we have determined the crystal structure of DBL6ε from the FCR3 parasite line and have compared it with the previously determined structure of that from the 3D7 line. We found significant differences particularly in the N-terminal region, which contains the first VB (VB1). Although DBL6ε is the most variable VAR2CSA domain, DBL6ε-FCR3 and DBL6ε-3D7 react with IgG purified from immune sera of pregnant women. Furthermore, IgG purified on one domain cross-reacts with the other, confirming the presence of cross-reactive epitopes. We also examined reactivity of immune sera to the four least variable VB (VB1, VB2, VB4 and VB5) using peptides with the consensus sequence closest, in turn, to the FCR3 or 3D7 domain. These results provide new molecular insights into immune escape by parasites expressing the VAR2CSA variant.
Assuntos
Antígenos de Protozoários/química , Malária Falciparum/imunologia , Malária Falciparum/metabolismo , Plasmodium falciparum/química , Complicações Parasitárias na Gravidez/imunologia , Proteínas de Protozoários/química , Sequência de Aminoácidos , Antígenos de Protozoários/imunologia , Cristalografia por Raios X , Feminino , Variação Genética/imunologia , Interações Hospedeiro-Parasita/imunologia , Humanos , Malária Falciparum/parasitologia , Dados de Sequência Molecular , Placenta/química , Placenta/imunologia , Placenta/parasitologia , Plasmodium falciparum/classificação , Plasmodium falciparum/imunologia , Gravidez , Complicações Parasitárias na Gravidez/metabolismo , Complicações Parasitárias na Gravidez/parasitologia , Estrutura Terciária de Proteína/genética , Proteínas de Protozoários/imunologiaRESUMO
Var2CSA, a key molecule linked with pregnancy-associated malaria (PAM), causes sequestration of Plasmodium falciparum infected erythrocytes (PEs) in the placenta by adhesion to chondroitin sulfate A (CSA). Var2CSA possesses a 300 kDa extracellular region composed of six Duffy-binding like (DBL) domains and a cysteine-rich interdomain region (CIDRpam) module. Although initial studies implicated several individual var2CSA DBL domains as important for adhesion of PEs to CSA, new studies revealed that these individual domains lack both the affinity and specificity displayed by the full-length extracellular region. Indeed, recent evidence suggests the presence of a single CSA-binding site formed by a higher-order domain organization rather than several independent binding sites located on the different domains. Here, we search for the minimal binding region within var2CSA that maintains high affinity and specificity for CSA binding, a characteristic feature of the full-length extracellular region. Accordingly, truncated recombinant var2CSA proteins comprising different domain combinations were expressed and their binding characteristics assessed against different sulfated glycosaminoglycans (GAGs). Our results indicate that the smallest region within var2CSA with similar binding properties to those of the full-length var2CSA is DBL1X-3X. We also demonstrate that inhibitory antibodies raised in rabbit against the full-length DBL1X-6ε target principally DBL3X and, to a lesser extent, DBL5ε. Taken together, our results indicate that efforts should focus on the DBL1X-3X region for developing vaccine and therapeutic strategies aimed at combating PAM.
Assuntos
Antígenos de Protozoários/metabolismo , Sulfatos de Condroitina/metabolismo , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Primers do DNA , Humanos , Reação em Cadeia da PolimeraseRESUMO
Pregnancy-associated malaria (PAM) is a serious consequence of sequestration of Plasmodium falciparum-parasitized erythrocytes (PE) in the placenta through adhesion to chondroitin sulfate A (CSA) present on placental proteoglycans. Recent work implicates var2CSA, a member of the PfEMP1 family, as the mediator of placental sequestration and as a key target for PAM vaccine development. Var2CSA is a 350 kDa transmembrane protein, whose extracellular region includes six Duffy-binding-like (DBL) domains. Due to its size and high cysteine content, the full-length var2CSA extracellular region has not hitherto been expressed in heterologous systems, thus limiting investigations to individual recombinant domains. Here we report for the first time the expression of the full-length var2CSA extracellular region (domains DBL1X to DBL6epsilon) from the 3D7 parasite strain using the human embryonic kidney 293 cell line. We show that the recombinant extracellular var2CSA region is correctly folded and that, unlike the individual DBL domains, it binds with high affinity and specificity to CSA (K(D) = 61 nM) and efficiently inhibits PE from binding to CSA. Structural characterization by analytical ultracentrifugation and small-angle x-ray scattering reveals a compact organization of the full-length protein, most likely governed by specific interdomain interactions, rather than an extended structure. Collectively, these data suggest that a high-affinity, CSA-specific binding site is formed by the higher-order structure of the var2CSA extracellular region. These results have important consequences for the development of an effective vaccine and therapeutic inhibitors.
Assuntos
Antígenos de Protozoários/química , Antígenos de Protozoários/metabolismo , Sulfatos de Condroitina/metabolismo , Espaço Extracelular/química , Plasmodium falciparum/metabolismo , Animais , Linhagem Celular , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Dicroísmo Circular , Decorina , Eritrócitos/metabolismo , Eritrócitos/parasitologia , Proteínas da Matriz Extracelular/metabolismo , Feminino , Humanos , Cinética , Modelos Moleculares , Parasitos/metabolismo , Placenta/metabolismo , Gravidez , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteoglicanas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
The use of carbohydrate-mimicking peptides to induce immune responses against surface polysaccharides of pathogenic bacteria offers a novel approach to vaccine development. Factors governing antigenic and immunogenic mimicry, however, are complex and poorly understood. We have addressed this question using the anti-lipopolysaccharide monoclonal antibody F22-4, which was raised against Shigella flexneri serotype 2a and shown to protect against homologous infection in a mouse model. In a previous crystallographic study, we described F22-4 in complex with two synthetic fragments of the O-antigen, the serotype-specific saccharide moiety of lipopolysaccharide. Here, we present a crystallographic and NMR study of the interaction of F22-4 with a dodecapeptide selected by phage display using the monoclonal antibody. Like the synthetic decasaccharide, the peptide binds to F22-4 with micromolar affinity. Although the peptide and decasaccharide use very similar regions of the antigen-binding site, indicating good antigenic mimicry, immunogenic mimicry by the peptide was not observed. The F22-4-antigen interaction is significantly more hydrophobic with the peptide than with oligosaccharides; nonetheless, all hydrogen bonds formed between the peptide and F22-4 have equivalents in the oligosaccharide complex. Two bridging water molecules are also in common, adding to partial structural mimicry. Whereas the bound peptide is entirely helical, its structure in solution, as shown by NMR, is helical in the central region only. Moreover, docking the NMR structure into the antigen-binding site shows that steric hindrance would occur, revealing poor complementarity between the major solution conformation and the antibody that could contribute to the absence of immunogenic mimicry.
Assuntos
Anticorpos , Mimetismo Molecular , Antígenos O , Peptídeos , Shigella flexneri/imunologia , Animais , Anticorpos/química , Anticorpos/imunologia , Sequência de Carboidratos , Cristalografia por Raios X , Camundongos , Conformação Molecular , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Antígenos O/química , Antígenos O/imunologia , Peptídeos/química , Peptídeos/imunologia , Conformação Proteica , TermodinâmicaRESUMO
Apical membrane antigen 1 (AMA1) has an important, but as yet uncharacterised, role in host cell invasion by the malaria parasite, Plasmodium. The protein, which is quite conserved between Plasmodium species, comprises an ectoplasmic region, a single transmembrane segment and a small cytoplasmic domain. The ectoplasmic region, which can induce protective immunity in animal models of human malaria, is a leading vaccine candidate that has entered clinical trials. The monoclonal antibody F8.12.19, raised against the recombinant ectoplasmic region of AMA1 from Plasmodium vivax, cross-reacts with homologues from Plasmodium knowlesi, Plasmodium cynomolgi, Plasmodium berghei and Plasmodium falciparum, as shown by immunofluorescence assays on mature schizonts. The binding of F8.12.19 to recombinant AMA1 from both P. vivax and P. falciparum was measured by surface plasmon resonance, revealing an apparent affinity constant that is about 100-fold weaker for the cross-reacting antigen when compared to the cognate antigen. Crystal structure analysis of Fab F8.12.19 complexed to AMA1 from P. vivax and P. falciparum shows that the monoclonal antibody recognises a discontinuous epitope located on domain III of the ectoplasmic region, the major component being a loop containing a cystine knot. The structures provide a basis for understanding the cross-reactivity. Antibody contacts are made mainly to main-chain and invariant side-chain atoms of AMA1; contact antigen residues that differ in sequence are located at the periphery of the antigen-binding site and can be accommodated at the interface between the two components of the complex. The implications for AMA1 vaccine development are discussed.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos de Protozoários/imunologia , Proteínas de Membrana/imunologia , Plasmodium vivax/imunologia , Proteínas de Protozoários/imunologia , Sequência de Aminoácidos , Animais , Antígenos de Protozoários/química , Antígenos de Protozoários/genética , Sequência de Bases , Reações Cruzadas , Cristalografia por Raios X , Cisteína/química , Epitopos , Técnica Indireta de Fluorescência para Anticorpo , Ligação de Hidrogênio , Proteínas de Membrana/química , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Dados de Sequência Molecular , Plasmodium vivax/química , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas de Protozoários/química , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Homologia de Sequência de Aminoácidos , Ressonância de Plasmônio de SuperfícieRESUMO
The monoclonal antibody 1696, elicited by HIV-1 protease, inhibits the activity of both HIV-1 and HIV-2 proteases with inhibition constants in the low nanomolar range. The antibody cross-reacts with peptides derived from the N-terminal region of both proteases. The crystal structure of the recombinant single-chain Fv fragment of 1696 complexed with an N-terminal peptide from the HIV-2 protease has been determined at 1.88A resolution. Interactions of the peptide with scFv1696 are compared with the previously reported structure of scFv1696 in complex with the corresponding peptide from HIV-1 protease. The origin of cross-reactivity of mAb1696 with HIV proteases is discussed.