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1.
PLoS Pathog ; 9(12): e1003840, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-24385910

RESUMEN

Malaria vaccine candidate Apical Membrane Antigen-1 (AMA1) induces protection, but only against parasite strains that are closely related to the vaccine. Overcoming the AMA1 diversity problem will require an understanding of the structural basis of cross-strain invasion inhibition. A vaccine containing four diverse allelic proteins 3D7, FVO, HB3 and W2mef (AMA1 Quadvax or QV) elicited polyclonal rabbit antibodies that similarly inhibited the invasion of four vaccine and 22 non-vaccine strains of P. falciparum. Comparing polyclonal anti-QV with antibodies against a strain-specific, monovalent, 3D7 AMA1 vaccine revealed that QV induced higher levels of broadly inhibitory antibodies which were associated with increased conserved face and domain-3 responses and reduced domain-2 response. Inhibitory monoclonal antibodies (mAb) raised against the QV reacted with a novel cross-reactive epitope at the rim of the hydrophobic trough on domain-1; this epitope mapped to the conserved face of AMA1 and it encompassed the 1e-loop. MAbs binding to the 1e-loop region (1B10, 4E8 and 4E11) were ∼10-fold more potent than previously characterized AMA1-inhibitory mAbs and a mode of action of these 1e-loop mAbs was the inhibition of AMA1 binding to its ligand RON2. Unlike the epitope of a previously characterized 3D7-specific mAb, 1F9, the 1e-loop inhibitory epitope was partially conserved across strains. Another novel mAb, 1E10, which bound to domain-3, was broadly inhibitory and it blocked the proteolytic processing of AMA1. By itself mAb 1E10 was weakly inhibitory but it synergized with a previously characterized, strain-transcending mAb, 4G2, which binds close to the hydrophobic trough on the conserved face and inhibits RON2 binding to AMA1. Novel inhibition susceptible regions and epitopes, identified here, can form the basis for improving the antigenic breadth and inhibitory response of AMA1 vaccines. Vaccination with a few diverse antigenic proteins could provide universal coverage by redirecting the immune response towards conserved epitopes.


Asunto(s)
Variación Antigénica , Antígenos de Protozoos/inmunología , Epítopos/inmunología , Vacunas contra la Malaria , Proteínas de la Membrana/inmunología , Plasmodium falciparum/inmunología , Proteínas Protozoarias/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Variación Antigénica/genética , Variación Antigénica/inmunología , Antígenos de Protozoos/química , Antígenos de Protozoos/genética , Células Cultivadas , Secuencia Conservada/inmunología , Mapeo Epitopo , Epítopos/genética , Inmunidad Humoral , Vacunas contra la Malaria/química , Vacunas contra la Malaria/inmunología , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Ratones , Ratones Desnudos , Modelos Moleculares , Plasmodium berghei/genética , Plasmodium berghei/inmunología , Estructura Terciaria de Proteína , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Conejos , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/inmunología
2.
Proc Natl Acad Sci U S A ; 108(32): 13275-80, 2011 Aug 09.
Artículo en Inglés | MEDLINE | ID: mdl-21788485

RESUMEN

The commitment of Plasmodium merozoites to invade red blood cells (RBCs) is marked by the formation of a junction between the merozoite and the RBC and the coordinated induction of the parasitophorous vacuole. Despite its importance, the molecular events underlying the parasite's commitment to invasion are not well understood. Here we show that the interaction of two parasite proteins, RON2 and AMA1, known to be critical for invasion, is essential to trigger junction formation. Using antibodies (Abs) that bind near the hydrophobic pocket of AMA1 and AMA1 mutated in the pocket, we identified RON2's binding site on AMA1. Abs specific for the AMA1 pocket blocked junction formation and the induction of the parasitophorous vacuole. We also identified the critical residues in the RON2 peptide (previously shown to bind AMA1) that are required for binding to the AMA1 pocket, namely, two conserved, disulfide-linked cysteines. The RON2 peptide blocked junction formation but, unlike the AMA1-specific Ab, did not block formation of the parasitophorous vacuole, indicating that formation of the junction and parasitophorous vacuole are molecularly distinct steps in the invasion process. Collectively, these results identify the binding of RON2 to the hydrophobic pocket of AMA1 as the step that commits Plasmodium merozoites to RBC invasion and point to RON2 as a potential vaccine candidate.


Asunto(s)
Merozoítos/metabolismo , Plasmodium falciparum/metabolismo , Plasmodium falciparum/patogenicidad , Proteínas Protozoarias/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Antiprotozoarios/inmunología , Sitios de Unión , Secuencia Conservada/genética , Cisteína/metabolismo , Citocalasina D/farmacología , Eritrocitos/efectos de los fármacos , Eritrocitos/parasitología , Fructosa-Bifosfato Aldolasa/química , Fructosa-Bifosfato Aldolasa/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas/efectos de los fármacos , Merozoítos/efectos de los fármacos , Merozoítos/ultraestructura , Modelos Biológicos , Datos de Secuencia Molecular , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/ultraestructura , Unión Proteica/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Proteínas Protozoarias/química , Relación Estructura-Actividad
3.
Infect Immun ; 78(2): 661-71, 2010 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-19948834

RESUMEN

Antibodies against apical membrane antigen 1 (AMA1) inhibit invasion of Plasmodium merozoites into red cells, and a large number of single nucleotide polymorphisms on AMA1 allow the parasite to escape inhibitory antibodies. The availability of a crystal structure makes it possible to test protein engineering strategies to develop a monovalent broadly reactive vaccine. Previously, we showed that a linear stretch of polymorphic residues (amino acids 187 to 207), localized within the C1 cluster on domain 1, conferred the highest level of escape from inhibitory antibodies, and these were termed antigenic escape residues (AER). Here we test the hypothesis that immunodampening the C1 AER will divert the immune system toward more conserved regions. We substituted seven C1 AER of the FVO strain Plasmodium falciparum AMA1 with alanine residues (ALA). The resulting ALA protein was less immunogenic than the native protein in rabbits. Anti-ALA antibodies contained a higher proportion of cross-reactive domain 2 and domain 3 antibodies and had higher avidity than anti-FVO. No overall enhancement of cross-reactive inhibitory activity was observed when anti-FVO and anti-ALA sera were compared for their ability to inhibit invasion. Alanine mutations at the C1 AER had shifted the immune response toward cross-strain-reactive epitopes that were noninhibitory, refuting the hypothesis but confirming the importance of the C1 cluster as an inhibitory epitope. We further demonstrate that naturally occurring polymorphisms that fall within the C1 cluster can predict escape from cross-strain invasion inhibition, reinforcing the importance of the C1 cluster genotype for antigenic categorization and allelic shift analyses in future phase 2b trials.


Asunto(s)
Antígenos de Protozoos/inmunología , Proteínas de la Membrana/inmunología , Proteínas Protozoarias/inmunología , Alanina , Animales , Anticuerpos Antiprotozoarios/inmunología , Especificidad de Anticuerpos , Antígenos de Protozoos/química , Western Blotting , Reacciones Cruzadas , Ensayo de Inmunoadsorción Enzimática , Vacunas contra la Malaria/inmunología , Proteínas de la Membrana/química , Mutagénesis Sitio-Dirigida , Reacción en Cadena de la Polimerasa , Estructura Cuaternaria de Proteína , Proteínas Protozoarias/química , Conejos
4.
Infect Immun ; 78(11): 4613-24, 2010 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-20823210

RESUMEN

The multiple antigen peptide (MAP) approach is an effective method to chemically synthesize and deliver multiple T-cell and B-cell epitopes as the constituents of a single immunogen. Here we report on the design, chemical synthesis, and immunogenicity of three Plasmodium falciparum MAP vaccines that incorporated antigenic epitopes from the sporozoite, liver, and blood stages of the life cycle. Antibody and cellular responses were determined in three inbred (C57BL/6, BALB/c, and A/J) strains, one congenic (HLA-A2 on the C57BL/6 background) strain, and one outbred strain (CD1) of mice. All three MAPs were immunogenic and induced both antibody and cellular responses, albeit in a somewhat genetically restricted manner. Antibodies against MAP-1, MAP-2, and MAP-3 had an antiparasite effect that was also dependent on the mouse major histocompatibility complex background. Anti-MAP-1 (CSP-based) antibodies blocked the invasion of HepG2 liver cells by P. falciparum sporozoites (highest, 95.16% in HLA-A2 C57BL/6; lowest, 11.21% in BALB/c). Furthermore, antibodies generated following immunizations with the MAP-2 (PfCSP, PfLSA-1, PfMSP-1(42), and PfMSP-3b) and MAP-3 (PfRAP-1, PfRAP-2, PfSERA, and PfMSP-1(42)) vaccines were able to reduce the growth of blood stage parasites in erythrocyte cultures to various degrees. Thus, MAP-based vaccines remain a viable option to induce effective antibody and cellular responses. These results warrant further development and preclinical and clinical testing of the next generation of candidate MAP vaccines that are based on the conserved protective epitopes from Plasmodium antigens that are widely recognized by populations of divergent HLA types from around the world.


Asunto(s)
Antígenos de Protozoos , Vacunas contra la Malaria , Malaria Falciparum/prevención & control , Vacunas de Subunidad , Vacunas Sintéticas , Secuencia de Aminoácidos , Animales , Animales no Consanguíneos , Anticuerpos Antiprotozoarios/sangre , Antígenos de Protozoos/química , Antígenos de Protozoos/inmunología , Línea Celular , Línea Celular Tumoral , Células Cultivadas , Diseño de Fármacos , Epítopos de Linfocito B/química , Epítopos de Linfocito B/inmunología , Epítopos de Linfocito T/química , Epítopos de Linfocito T/inmunología , Eritrocitos/parasitología , Femenino , Antígeno HLA-A2/genética , Antígeno HLA-A2/metabolismo , Hepatocitos/parasitología , Humanos , Inmunización , Vacunas contra la Malaria/administración & dosificación , Vacunas contra la Malaria/síntesis química , Vacunas contra la Malaria/química , Vacunas contra la Malaria/inmunología , Malaria Falciparum/inmunología , Ratones , Ratones Congénicos , Ratones Endogámicos , Datos de Secuencia Molecular , Plasmodium falciparum/inmunología , Plasmodium falciparum/patogenicidad , Linfocitos T/inmunología , Vacunas de Subunidad/administración & dosificación , Vacunas de Subunidad/síntesis química , Vacunas de Subunidad/química , Vacunas de Subunidad/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/química , Vacunas Sintéticas/inmunología
5.
Sci Rep ; 7(1): 17146, 2017 12 07.
Artículo en Inglés | MEDLINE | ID: mdl-29215067

RESUMEN

The symptoms of malaria are brought about by blood-stage parasites, which are established when merozoites invade human erythrocytes. Our understanding of the molecular events that underpin erythrocyte invasion remains hampered by the short-period of time that merozoites are invasive. To address this challenge, a Plasmodium falciparum gamma-irradiated long-lived merozoite (LLM) line was developed and investigated. Purified LLMs invaded erythrocytes by an increase of 10-300 fold compared to wild-type (WT) merozoites. Using an integrated omics approach, we investigated the basis for the phenotypic difference. Only a few single nucleotide polymorphisms within the P. falciparum genome were identified and only marginal differences were observed in the merozoite transcriptomes. By contrast, using label-free quantitative mass-spectrometry, a significant change in protein abundance was noted, of which 200 were proteins of unknown function. We determined the relative molar abundance of over 1100 proteins in LLMs and further characterized the major merozoite surface protein complex. A unique processed MSP1 intermediate was identified in LLM but not observed in WT suggesting that delayed processing may be important for the observed phenotype. This integrated approach has demonstrated the significant role of the merozoite proteome during erythrocyte invasion, while identifying numerous unknown proteins likely to be involved in invasion.


Asunto(s)
Eritrocitos/metabolismo , Malaria Falciparum/metabolismo , Merozoítos/metabolismo , Plasmodium falciparum/metabolismo , Proteoma , Proteínas Protozoarias/metabolismo , Transcriptoma , Animales , Eritrocitos/parasitología , Humanos , Malaria Falciparum/genética , Malaria Falciparum/parasitología , Merozoítos/genética , Plasmodium falciparum/genética , Plasmodium falciparum/aislamiento & purificación , Proteínas Protozoarias/genética
6.
Nucleic Acids Res ; 30(10): 2224-31, 2002 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-12000842

RESUMEN

Plasmodium falciparum intraerythrocytic development is a complex process. Development proceeds rapidly from the trophozoite phase of nutrient acquisition and growth through to the synthetic and reproductive schizont phase, which ends with production of new invasive merozoites. During this process, the malaria parasite must express a series of different gene products, depending on its metabolic and synthetic needs. We are particularly interested in the development of the merozoite's organelles in the apical complex, which form during the later schizont stages. We have used quantitative real-time RT-PCR fluorogenic 5' nuclease assays (TaqMan) for the first time on malaria parasites for analysis of erythrocytic stage-specific gene expression. We analyzed transcripts of the P.falciparum eba-175 and other erythrocyte binding-like (ebl) family genes in temperature-synchronized parasites and found ebl genes have tightly controlled, stage-specific transcription. As expected, eba-175 transcripts were abundant only at the end of schizont development in a pattern most common among ebl, including baebl, pebl and jesebl. The maebl transcript pattern was unique, peaking at mid-late trophozoite stage, but absent in late-stage schizonts. ebl-1 demonstrated another pattern of expression, which peaked during mid-schizont stage and then significantly diminished in late-stage schizonts. Our analysis demonstrates that using real-time RT-PCR fluorogenic 5' nuclease assays is a sensitive, quantitative method for analysis of Plasmodium transcripts.


Asunto(s)
Antígenos de Protozoos , Genes Protozoarios/genética , Plasmodium falciparum/genética , Proteínas Protozoarias , Animales , Southern Blotting , Proteínas Portadoras/genética , ADN Protozoario/genética , Regulación del Desarrollo de la Expresión Génica , Plasmodium falciparum/crecimiento & desarrollo , Reacción en Cadena de la Polimerasa/métodos , ARN Protozoario/genética , ARN Protozoario/metabolismo , Receptores de Superficie Celular/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Temperatura , Factores de Tiempo , Transcripción Genética
7.
Mol Biochem Parasitol ; 128(2): 195-204, 2003 May.
Artículo en Inglés | MEDLINE | ID: mdl-12742586

RESUMEN

Merozoite Surface Protein-1(42) (MSP-1(42)) is a leading vaccine candidate against erythrocytic malaria parasites. We cloned and expressed Plasmodium falciparum MSP-1(42) (3D7 clone) in Escherichia coli. The antigen was purified to greater than 95% homogeneity by using nickel-, Q- and carboxy-methyl (CM)-substituted resins. The final product, designated Falciparum Merozoite Protein-1 (FMP1), had endotoxin levels significantly lower than FDA standards. It was structurally correct based on binding conformation-dependent mAbs, and was stable. Functional antibodies from rabbits vaccinated with FMP1 in Freund's adjuvant inhibited parasite growth in vitro and also inhibited secondary processing of MSP-1(42). FMP1 formulated with GlaxoSmithKline Biologicals (GSK) adjuvant, AS02A or alum was safe and immunogenic in rhesus (Macaca mulatta) monkeys.


Asunto(s)
Vacunas contra la Malaria/inmunología , Proteína 1 de Superficie de Merozoito/inmunología , Plasmodium falciparum/inmunología , Animales , Anticuerpos Antiprotozoarios/sangre , Evaluación Preclínica de Medicamentos , Femenino , Macaca mulatta , Vacunas contra la Malaria/química , Vacunas contra la Malaria/genética , Malaria Falciparum/prevención & control , Masculino , Proteína 1 de Superficie de Merozoito/clasificación , Modelos Genéticos , Datos de Secuencia Molecular , Plasmodium falciparum/crecimiento & desarrollo , Conejos , Vacunas Sintéticas/química , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología
8.
Methods Mol Med ; 72: 535-54, 2002.
Artículo en Inglés | MEDLINE | ID: mdl-12125152

RESUMEN

Erythrocytic cycle malaria parasite growth or invasion inhibition assays (GIA) compare the effects of various test and control substances on malaria parasite growth in erythrocytes or invasion into erythrocytes in vitro. Although inhibitions by antimalarial drugs in vitro correlate well with drug protective levels required in vivo, as yet there are too few data to know how well inhibitions by antibodies in vitro correlate with the types and degrees of immune protection in vivo. Antibody-mediated GIA is frequently complicated by parasite strain-specific inhibitions, as well as nonspecific inhibitory factors generated in sera collected or stored under nonoptimal conditions. In this chapter, we describe methods for collecting and processing sera, for using different strains of parasite, and a simplified method for staining parasite DNA with Hoechst dye 33342 before quantitating parasites using ultraviolet (UV)-excited flow cytometry. We also describe a new type of GIA using suspension cultures in a 48-well plate. Critical to this method is enclosing the plate in a gassed, heat-sealed plastic bag, which, being low mass, can easily be rested at a 13.5 degrees angle on a rotor platform (114 rpm with 1-in. displacement) to produce gentle pulsatile waves of media in each well. The suspension GIA, which, relative to the static GIA, increased inhibition by one antibody and decreased inhibition by another (Table 1), may better simulate in vivo blood flow and may thus better predict in vivo efficacy.


Asunto(s)
Eritrocitos/parasitología , Malaria/fisiopatología , Plasmodium/crecimiento & desarrollo , Animales , Humanos , Estadios del Ciclo de Vida , Malaria/parasitología , Malaria Falciparum/parasitología , Malaria Falciparum/fisiopatología , Parasitología/métodos , Plasmodium/patogenicidad , Plasmodium falciparum/crecimiento & desarrollo , Plasmodium falciparum/parasitología , Especificidad de la Especie , Virulencia
9.
Nat Commun ; 4: 2261, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23907321

RESUMEN

Plasmodium falciparum resistance to artemisinin derivatives, the first-line antimalarial drug, drives the search for new classes of chemotherapeutic agents. Current discovery is primarily directed against the intracellular forms of the parasite. However, late schizont-infected red blood cells (RBCs) may still rupture and cause disease by sequestration; consequently targeting invasion may reduce disease severity. Merozoite invasion of RBCs requires interaction between two parasite proteins AMA1 and RON2. Here we identify the first inhibitor of this interaction that also blocks merozoite invasion in genetically distinct parasites by screening a library of over 21,000 compounds. We demonstrate that this inhibition is mediated by the small molecule binding to AMA1 and blocking the formation of AMA1-RON complex. Electron microscopy confirms that the inhibitor prevents junction formation, a critical step in invasion that results from AMA1-RON2 binding. This study uncovers a strategy that will allow for highly effective combination therapies alongside existing antimalarial drugs.


Asunto(s)
Eritrocitos/parasitología , Malaria/parasitología , Parásitos/metabolismo , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Antimaláricos/análisis , Antimaláricos/química , Antimaláricos/farmacología , Artemisininas/farmacología , Eritrocitos/efectos de los fármacos , Eritrocitos/ultraestructura , Humanos , Concentración 50 Inhibidora , Merozoítos/efectos de los fármacos , Merozoítos/ultraestructura , Parásitos/efectos de los fármacos , Plasmodium falciparum/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Bibliotecas de Moléculas Pequeñas/análisis , Bibliotecas de Moléculas Pequeñas/química , Familia-src Quinasas/antagonistas & inhibidores , Familia-src Quinasas/metabolismo
10.
Mol Biochem Parasitol ; 177(1): 57-60, 2011 May.
Artículo en Inglés | MEDLINE | ID: mdl-21251929

RESUMEN

A majority of Plasmodium falciparum strains invade erythrocytes through interactions with sialic acid (SA) on glycophorins. However, we recently reported that complement receptor 1 (CR1) is a SA-independent invasion receptor of many laboratory strains of P. falciparum. To determine the role of CR1 in erythrocyte invasion among P. falciparum field isolates, we tested eight isolates obtained from children in Kenya. All the parasites examined were capable of invading in a SA-independent manner, and invasion of neuraminidase-treated erythrocytes was nearly completely blocked by anti-CR1 and soluble CR1 (sCR1). In addition, anti-CR1 and sCR1 partially inhibited invasion of intact erythrocytes in a majority of isolates tested. Sequencing of the hypervariable region of P. falciparum AMA-1 showed considerable diversity among all the isolates. These data demonstrate that CR1 mediates SA-independent erythrocyte invasion in P. falciparum field isolates.


Asunto(s)
Eritrocitos/metabolismo , Malaria Falciparum/metabolismo , Plasmodium falciparum/fisiología , Receptores de Complemento 3b/metabolismo , Secuencia de Aminoácidos , Antígenos de Protozoos/genética , Niño , Eritrocitos/parasitología , Femenino , Interacciones Huésped-Parásitos , Humanos , Malaria Falciparum/parasitología , Masculino , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Filogenia , Plasmodium falciparum/clasificación , Plasmodium falciparum/genética , Plasmodium falciparum/aislamiento & purificación , Unión Proteica , Proteínas Protozoarias/genética , Alineación de Secuencia
11.
PLoS One ; 6(4): e18393, 2011 Apr 14.
Artículo en Inglés | MEDLINE | ID: mdl-21533224

RESUMEN

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 (IC(50) 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 IC(50) 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.


Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígenos de Protozoos/metabolismo , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/metabolismo , Animales , Antígenos de Protozoos/inmunología , Plasmodium falciparum/inmunología , Plasmodium falciparum/fisiología , Proteínas Protozoarias/inmunología
13.
PLoS One ; 4(12): e8138, 2009 Dec 03.
Artículo en Inglés | MEDLINE | ID: mdl-19997632

RESUMEN

A Plasmodium falciparum 3D7 strain Apical Membrane Antigen-1 (AMA1) vaccine, formulated with AS02(A) adjuvant, slowed parasite growth in a recent Phase 1/2a trial, however sterile protection was not observed. We tested this AS02(A), and a Montanide ISA720 (ISA) formulation of 3D7 AMA1 in Aotus monkeys. The 3D7 parasite does not invade Aotus erythrocytes, hence two heterologous strains, FCH/4 and FVO, were used for challenge, FCH/4 AMA1 being more homologous to 3D7 than FVO AMA1. Following three vaccinations, the monkeys were challenged with 50,000 FCH/4 or 10,000 FVO parasites. Three of the six animals in the AMA+ISA group were protected against FCH/4 challenge. One monkey did not become parasitemic, another showed only a short period of low level parasitemia that self-cured, and a third animal showed a delay before exhibiting its parasitemic phase. This is the first protection shown in primates with a recombinant P. falciparum AMA1 without formulation in Freund's complete adjuvant. No animals in the AMA+AS02(A) group were protected, but this group exhibited a trend towards reduced growth rate. A second group of monkeys vaccinated with AMA+ISA vaccine was not protected against FVO challenge, suggesting strain-specificity of AMA1-based protection. Protection against FCH/4 strain correlated with the quantity of induced antibodies, as the protected animals were the only ones to have in vitro parasite growth inhibitory activity of >70% at 1:10 serum dilution; immuno-fluorescence titers >8,000; ELISA titers against full-length AMA1 >300,000 and ELISA titer against AMA1 domains1+2 >100,000. A negative correlation between log ELISA titer and day 11 cumulative parasitemia (Spearman rank r = -0.780, p value = 0.0001), further confirmed the relationship between antibody titer and protection. High titers of cross-strain inhibitory antibodies against AMA1 are therefore critical to confer solid protection, and the Aotus model can be used to down-select future AMA1 formulations, prior to advanced human trials.


Asunto(s)
Anticuerpos Antiprotozoarios/inmunología , Malaria Falciparum/inmunología , Malaria Falciparum/prevención & control , Proteínas Protozoarias/inmunología , Secuencia de Aminoácidos , Animales , Formación de Anticuerpos/inmunología , Especificidad de Anticuerpos/inmunología , Aotus trivirgatus , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Inmunoensayo , Vacunas contra la Malaria/química , Vacunas contra la Malaria/inmunología , Malaria Falciparum/complicaciones , Datos de Secuencia Molecular , Parasitemia/complicaciones , Parasitemia/inmunología , Plasmodium falciparum/crecimiento & desarrollo , Plasmodium falciparum/inmunología , Pliegue de Proteína , Proteínas Protozoarias/química , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Análisis de Secuencia de Proteína , Volumetría , Vacunación
14.
Infect Immun ; 75(4): 2012-25, 2007 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-17283083

RESUMEN

Intermittent episodes of febrile illness are the most benign and recognized symptom of infection with malaria parasites, although the effects on parasite survival and virulence remain unclear. In this study, we identified the molecular factors altered in response to febrile temperature by measuring differential expression levels of individual genes using high-density oligonucleotide microarray technology and by performing biological assays in asexual-stage Plasmodium falciparum parasite cultures incubated at 37 degrees C and 41 degrees C (an elevated temperature that is equivalent to malaria-induced febrile illness in the host). Elevated temperature had a profound influence on expression of individual genes; 336 of approximately 5,300 genes (6.3% of the genome) had altered expression profiles. Of these, 163 genes (49%) were upregulated by twofold or greater, and 173 genes (51%) were downregulated by twofold or greater. In-depth sensitive sequence profile analysis revealed that febrile temperature-induced responses caused significant alterations in the major parasite biologic networks and pathways and that these changes are well coordinated and intricately linked. One of the most notable transcriptional changes occurs in genes encoding proteins containing the predicted Pexel motifs that are exported into the host cytoplasm or inserted into the host cell membrane and are likely to be associated with erythrocyte remodeling and parasite sequestration functions. Using our sensitive computational analysis, we were also able to assign biochemical or biologic functional predictions for at least 100 distinct genes previously annotated as "hypothetical." We find that cultivation of P. falciparum parasites at 41 degrees C leads to parasite death in a time-dependent manner. The presence of the "crisis forms" and the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling-positive parasites following heat treatment strongly support the notion that an apoptosis-like cell death mechanism might be induced in response to febrile temperatures. These studies enhance the possibility of designing vaccines and drugs on the basis of disruption in molecules and pathways of parasite survival and virulence activated in response to febrile temperatures.


Asunto(s)
Eritrocitos/parasitología , Regulación de la Expresión Génica , Genes Protozoarios , Plasmodium falciparum/genética , Plasmodium falciparum/fisiología , Temperatura , Adaptación Fisiológica , Animales , Apoptosis , Perfilación de la Expresión Génica , Respuesta al Choque Térmico , Etiquetado Corte-Fin in Situ , Redes y Vías Metabólicas , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/fisiología , ARN Mensajero/análisis , ARN Mensajero/genética , ARN Protozoario/análisis , ARN Protozoario/genética , Transcripción Genética
15.
Infect Immun ; 73(4): 2116-22, 2005 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-15784553

RESUMEN

Antibodies against apical membrane antigen 1 (AMA-1) of Plasmodium falciparum inhibit merozoite invasion into erythrocytes. Invasion-inhibitory polyclonal AMA-1 antibodies inhibit secondary proteolytic processing and surface redistribution of AMA-1 on merozoites. We present evidence supporting inhibition of processing and redistribution as probable causes of inhibition of invasion by polyclonal antibodies. Polyclonal anti-AMA-1 was much more inhibitory than monoclonal antibody (MAb) 4G2dc1 in an invasion assay. Although both polyclonal and monoclonal immunoglobulin G (IgG) inhibited secondary processing of the 66-kDa form of AMA-1, only polyclonal IgG caused its anomalous processing, inhibited its redistribution, and cross-linked soluble forms of AMA-1 on merozoites. Moreover, Fab fragments of polyclonal IgG that fail to cross-link did not show the enhancement of inhibitory effect over intact IgG, as observed in the case of Fab fragments of MAb 4G2dc1. We propose that although blocking of biologically important sites is a common direct mode of action of anti-AMA-1 antibodies, blocking of AMA-1 secondary processing and redistribution are additional indirect inhibitory mechanisms by which polyclonal IgG inhibits invasion. We also report a processing inhibition assay that uses a C-terminal AMA-1-specific MAb, 28G2dc1, to detect merozoite-bound remnants of processing (approximately 20 kDa from normal processing to 48 and 44 kDa and approximately 10 kDa from anomalous processing to a 52-kDa soluble form of AMA-1). The ratio of intensity of 10-kDa bands to the sum of 10- and 20-kDa bands was positively correlated with inhibition of invasion by polyclonal antibodies. This assay may serve as an important immunochemical correlate for inhibition of invasion.


Asunto(s)
Anticuerpos Antiprotozoarios/farmacología , Antígenos de Protozoos/inmunología , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/inmunología , Plasmodium falciparum/inmunología , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/inmunología , Animales , Anticuerpos Monoclonales/farmacología , Antígenos de Protozoos/metabolismo , Eritrocitos/parasitología , Sueros Inmunes/farmacología , Fragmentos Fab de Inmunoglobulinas/farmacología , Inmunoglobulina G/farmacología , Proteínas de la Membrana/metabolismo , Ratones , Proteínas Protozoarias/metabolismo , Conejos
16.
Proc Natl Acad Sci U S A ; 100(21): 12295-300, 2003 Oct 14.
Artículo en Inglés | MEDLINE | ID: mdl-14526103

RESUMEN

Apical membrane antigen 1 (AMA-1) is a promising vaccine candidate for Plasmodium falciparum malaria. Antibodies against AMA-1 of P. falciparum (PfAMA-1) interrupt merozoite invasion into RBCs. Initially localized within the apical complex, PfAMA-1 is proteolytically processed and redistributed circumferentially on merozoites at about the time of their release and invasion into RBCs. An 83-kDa precursor form of PfAMA-1 is processed to 66-kDa and then to 48- and 44-kDa products. We show that, even at low concentrations, IgG antibodies against correctly folded recombinant PfAMA-1 cross-linked and trapped the 52-, 48-, and 44-kDa proteolytic products on merozoites. These products are normally shed into the culture medium. At higher concentrations antibodies inhibited invasion into RBCs and caused a reduction in the amount of 44- and 48-kDa products, both on merozoites and in the culture medium. A corresponding increase also occurred in the amount of the 66- and 52-kDa forms detected on the merozoites. These antibodies also prevented circumferential redistribution of AMA-1. In contrast, monovalent invasion-inhibitory Fab fragments caused accumulation of 66- and 52-kDa forms, with no cross-linking, trapping, or prevention of redistribution. Antibodies at low concentrations can be used as trapping agents for intermediate and soluble forms of AMA-1 and are useful for studying proteolytic processing of AMA-1. With this technique, it was confirmed that protease inhibitor chymostatin and Ca2+ chelators can inhibit the breakdown of the 66-kDa form. We propose that antibodies to AMA-1 capable of inhibiting erythrocyte invasion act by disrupting proteolytic processing of AMA-1.


Asunto(s)
Anticuerpos Antiprotozoarios/metabolismo , Antígenos de Protozoos/inmunología , Antígenos de Protozoos/metabolismo , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/metabolismo , Plasmodium falciparum/inmunología , Proteínas Protozoarias/inmunología , Proteínas Protozoarias/metabolismo , Animales , Presentación de Antígeno/efectos de los fármacos , Quelantes/farmacología , Técnicas In Vitro , Vacunas contra la Malaria/inmunología , Vacunas contra la Malaria/metabolismo , Modelos Inmunológicos , Plasmodium falciparum/crecimiento & desarrollo , Plasmodium falciparum/metabolismo , Inhibidores de Proteasas/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Conejos , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo
17.
Infect Immun ; 72(8): 4464-70, 2004 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-15271904

RESUMEN

The apical membrane antigen 1 of Plasmodium falciparum is one of the leading candidate antigens being developed as a vaccine to prevent malaria. This merozoite transmembrane protein has an ectodomain that can be divided into three subdomains (D I, D II, and D III). We have previously expressed a major portion of this ectodomain and have shown that it can induce antibodies that prevent merozoite invasion into red blood cells in an in vitro growth and invasion assay. To analyze the antibody responses directed against the individual subdomains, we constructed six different genes that express each of the domains separately (D I, D II, or D III) or in combination with another domain (D I+II, D II+III, or D I+III). These proteins were purified and used to immunize rabbits to raise construct-specific antibodies. We demonstrated that D I+II induced a significant amount of the growth-inhibitory antibodies active in the growth and invasion assay.


Asunto(s)
Anticuerpos Antiprotozoarios/sangre , Antígenos de Protozoos/química , Antígenos de Protozoos/inmunología , Proteínas de la Membrana/química , Proteínas de la Membrana/inmunología , Plasmodium falciparum/inmunología , Plasmodium falciparum/patogenicidad , Proteínas Protozoarias/química , Proteínas Protozoarias/inmunología , Animales , Anticuerpos Antiprotozoarios/inmunología , Antígenos de Protozoos/genética , Antígenos de Protozoos/metabolismo , Eritrocitos/parasitología , Escherichia coli/genética , Escherichia coli/metabolismo , Inmunización , Vacunas contra la Malaria/administración & dosificación , Vacunas contra la Malaria/inmunología , Malaria Falciparum/prevención & control , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Plasmodium falciparum/química , Plasmodium falciparum/crecimiento & desarrollo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Conejos , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo
18.
Science ; 301(5639): 1503-8, 2003 Sep 12.
Artículo en Inglés | MEDLINE | ID: mdl-12893887

RESUMEN

The completion of the genome sequence for Plasmodium falciparum, the species responsible for most malaria human deaths, has the potential to reveal hundreds of new drug targets and proteins involved in pathogenesis. However, only approximately 35% of the genes code for proteins with an identifiable function. The absence of routine genetic tools for studying Plasmodium parasites suggests that this number is unlikely to change quickly if conventional serial methods are used to characterize encoded proteins. Here, we use a high-density oligonucleotide array to generate expression profiles of human and mosquito stages of the malaria parasite's life cycle. Genes with highly correlated levels and temporal patterns of expression were often involved in similar functions or cellular processes.


Asunto(s)
Perfilación de la Expresión Génica , Expresión Génica , Genes Protozoarios , Plasmodium falciparum/crecimiento & desarrollo , Plasmodium falciparum/genética , Animales , Anopheles/parasitología , Ciclo Celular , Cromosomas/genética , Análisis por Conglomerados , Eritrocitos/parasitología , Regulación del Desarrollo de la Expresión Génica , Humanos , Estadios del Ciclo de Vida , Hígado/parasitología , Malaria Falciparum/parasitología , Análisis de Secuencia por Matrices de Oligonucleótidos , Plasmodium falciparum/metabolismo , Proteoma , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/fisiología , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Protozoario/genética , ARN Protozoario/metabolismo , Glándulas Salivales/parasitología , Esporozoítos/genética , Esporozoítos/crecimiento & desarrollo , Transcripción Genética
19.
Infect Immun ; 70(6): 3101-10, 2002 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-12011004

RESUMEN

The apical membrane antigen 1 (AMA1) has emerged as a promising vaccine candidate against malaria. Advanced evaluation of its protective efficacy in humans requires the production of highly purified and correctly folded protein. We describe here a process for the expression, fermentation, refolding, and purification of the recombinant ectodomain of AMA1 (amino acids 83(Gly) to 531(Glu)) of Plasmodium falciparum (3D7) produced in Escherichia coli. A synthetic gene containing an E. coli codon bias was cloned into a modified pET32 plasmid, and the recombinant protein was produced by using a redox-modified E. coli strain, Origami (DE3). A purification process was developed that included Sarkosyl extraction followed by affinity purification on a Ni-nitrilotriacetic acid column. The recombinant AMA1 was refolded in the presence of reduced and oxidized glutathione and further purified by using two ion-exchange chromatographic steps. The final product, designated AMA1/E, was homogeneous, monomeric, and >99% pure and had low endotoxin content and low host cell contamination. Analysis of AMA1/E showed that it had the predicted primary sequence, and tertiary structure analysis confirmed its compact disulfide-bonded nature. Rabbit antibodies made to the protein recognized the native parasite AMA1 and inhibited the growth of the P. falciparum homologous 3D7 clone in an in vitro assay. Reduction-sensitive epitopes on AMA1/E were shown to be necessary for the production of inhibitory anti-AMA1 antibodies. AMA1/E was recognized by a conformation-dependent, growth-inhibitory monoclonal antibody, 4G2dc1. The process described here was successfully scaled up to produce AMA1/E protein under GMP conditions, and the product was found to induce highly inhibitory antibodies in rabbits.


Asunto(s)
Antígenos de Protozoos/inmunología , Proteínas de la Membrana/inmunología , Plasmodium falciparum/inmunología , Pliegue de Proteína , Proteínas Protozoarias/inmunología , Sarcosina/análogos & derivados , Animales , Anticuerpos Antiprotozoarios/inmunología , Antígenos de Protozoos/biosíntesis , Antígenos de Protozoos/genética , Antígenos de Protozoos/aislamiento & purificación , Cromatografía de Afinidad/métodos , Cromatografía por Intercambio Iónico/métodos , Cisteína , Ácido Edético , Endotoxinas , Escherichia coli , Fermentación , Expresión Génica , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Proteínas de la Membrana/aislamiento & purificación , Níquel , Plasmodium falciparum/genética , Estructura Terciaria de Proteína , Proteínas Protozoarias/biosíntesis , Proteínas Protozoarias/genética , Proteínas Protozoarias/aislamiento & purificación , Conejos , Sacarosa
20.
Nature ; 419(6906): 520-6, 2002 Oct 03.
Artículo en Inglés | MEDLINE | ID: mdl-12368866

RESUMEN

The completion of the Plasmodium falciparum clone 3D7 genome provides a basis on which to conduct comparative proteomics studies of this human pathogen. Here, we applied a high-throughput proteomics approach to identify new potential drug and vaccine targets and to better understand the biology of this complex protozoan parasite. We characterized four stages of the parasite life cycle (sporozoites, merozoites, trophozoites and gametocytes) by multidimensional protein identification technology. Functional profiling of over 2,400 proteins agreed with the physiology of each stage. Unexpectedly, the antigenically variant proteins of var and rif genes, defined as molecules on the surface of infected erythrocytes, were also largely expressed in sporozoites. The detection of chromosomal clusters encoding co-expressed proteins suggested a potential mechanism for controlling gene expression.


Asunto(s)
Estadios del Ciclo de Vida , Plasmodium falciparum/crecimiento & desarrollo , Proteoma , Proteínas Protozoarias/fisiología , Animales , Antimaláricos/farmacología , Cromosomas , Eritrocitos/parasitología , Femenino , Genoma de Protozoos , Células Germinativas , Humanos , Vacunas contra la Malaria , Masculino , Plasmodium falciparum/genética , Plasmodium falciparum/fisiología , Proteínas Protozoarias/genética
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