RESUMEN
Apical membrane antigen 1 (AMA1) is currently one of the leading malarial vaccine candidates. Anti-AMA1 antibodies can inhibit the invasion of erythrocytes by Plasmodium merozoites and prevent the multiplication of blood-stage parasites. Here we describe an anti-AMA1 monoclonal antibody (MAb 1F9) that inhibits the invasion of Plasmodium falciparum parasites in vitro. We show that both reactivity of MAb 1F9 with AMA1 and MAb 1F9-mediated invasion inhibition were strain specific. Site-directed mutagenesis of a fragment of AMA1 displayed on M13 bacteriophage identified a single polymorphic residue in domain I of AMA1 that is critical for MAb 1F9 binding. The identities of all other polymorphic residues investigated in this domain had little effect on the binding of the antibody. Examination of the P. falciparum AMA1 crystal structure localized this residue to a surface-exposed alpha-helix at the apex of the polypeptide. This description of a polymorphic inhibitory epitope on AMA1 adds supporting evidence to the hypothesis that immune pressure is responsible for the polymorphisms seen in this molecule.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Antiprotozoarios/inmunología , Antígenos de Protozoos/inmunología , Mapeo Epitopo , Proteínas de la Membrana/inmunología , Plasmodium falciparum/inmunología , Proteínas Protozoarias/inmunología , Secuencia de Aminoácidos , Animales , Antígenos de Protozoos/química , Eritrocitos/parasitología , Humanos , Proteínas de la Membrana/química , Datos de Secuencia Molecular , Conformación Proteica , Proteínas Protozoarias/química , Especificidad de la EspecieRESUMEN
We describe an approach for the rapid mapping of epitopes within a malaria antigen using a combination of phage display techniques. Phage display of antigen fragments identifies the location of the epitopes, then random peptide libraries displayed on phage are employed to identify accurately amino acids involved in the epitope. Finally, phage display of mutant fragments confirms the role of each residue in the epitope. This approach was applied to the apical membrane antigen-1 (AMA1), which is a leading candidate for inclusion in a vaccine directed against the asexual blood stages of Plasmodium falciparum. As part of the effort both to understand the function of AMA1 in the parasite life cycle and to define the specificity of protective immune responses, a panel of monoclonal antibodies (MAbs) was generated to obtain binding reagents to the various domains within the molecule. There is a pressing need to determine rapidly the regions recognized by these antibodies and the structural requirements required within AMA1 for high affinity binding of the MAbs. Using phage displaying random AMA1 fragments, it was shown that MAb5G8 recognizes a short linear epitope within the pro-domain of AMA1 whereas the epitope recognized by MAb 1F9 is reduction sensitive and resides within a disulphide-bonded 57 amino acid sub-domain of domain-1. Phage displaying random peptide libraries and mutant AMA1 fragments were employed for fine mapping of the MAb5G8 core epitope to a three-residue sequence in the AMA1 prodomain.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígenos de Protozoos/inmunología , Mapeo Epitopo/métodos , Proteínas de la Membrana/inmunología , Plasmodium falciparum/inmunología , Proteínas Protozoarias/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/genética , Especificidad de Anticuerpos , Antígenos de Protozoos/química , Antígenos de Protozoos/genética , Bacteriófagos/genética , Disulfuros/química , Epítopos/inmunología , Eritrocitos/parasitología , Proteínas de la Membrana/química , Datos de Secuencia Molecular , Mutación , Biblioteca de Péptidos , Péptidos/genética , Péptidos/inmunología , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Protozoarias/química , Alineación de SecuenciaRESUMEN
We describe an unusual tryptophan-rich protein of Plasmodium falciparum that contains threonine-rich repeats. The protein is encoded by a 2.5 kb gene with a two-exon structure including a short AT-rich intron that is spliced out of the mature message. The 5' end of the gene encodes a hydrophobic region, which is assumed to be a signal peptide. The peptide sequence is characterised by a tryptophan-rich region and a block of degenerate threonine repeats. The protein is synthesised throughout the asexual life cycle and has an apparent molecular weight of approximately 94 kDa. It has a variable molecular weight in different strains of P. falciparum. Length polymorphisms can be found in the intron region and the second exon. Four single nucleotide mutations are localised in the tryptophan-rich region and two were found in the threonine-repeat block. Homology searches based on gene structure and amino acid sequence revealed a relationship with a P. yoelii antigen that has been used successfully in vaccine studies. Thus, this P. falciparum antigen should be considered an additional candidate for assessment in vaccination against the asexual blood-stages of P. falciparum.
Asunto(s)
Vacunas contra la Malaria/genética , Proteínas de la Membrana/genética , Plasmodium falciparum/inmunología , Plasmodium yoelii/inmunología , Proteínas Protozoarias/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Malaria/prevención & control , Vacunas contra la Malaria/inmunología , Proteínas de la Membrana/química , Proteínas de la Membrana/inmunología , Datos de Secuencia Molecular , Proteínas Protozoarias/química , Proteínas Protozoarias/inmunología , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
Apical membrane antigen 1 (AMA1) is considered one of the leading candidates for inclusion in a vaccine against blood stages of Plasmodium falciparum. Although the ama1 gene is relatively conserved compared to those for some other potential vaccine components, numerous point mutations have resulted in amino acid substitutions at many sites in the polypeptide. The polymorphisms in AMA1 have been attributed to the diversifying selection pressure of the protective immune responses. It was therefore of interest to investigate the impact of sequence diversity in P. falciparum AMA1 on the ability of anti-AMA1 antibodies to inhibit the invasion of erythrocytes in vitro by P. falciparum merozoites. For these studies, we used antibodies to recombinant P. falciparum 3D7 AMA1 ectodomain, which was prepared for testing in early clinical trials. Antibodies were raised in rabbits to the antigen formulated in Montanide ISA720, and human antibodies to AMA1 were isolated by affinity purification from the plasma of adults living in regions of Papua New Guinea where malaria is endemic. Both rabbit and human anti-AMA1 antibodies were found to be strongly inhibitory to the invasion of erythrocytes by merozoites from both the homologous and two heterologous lines of P. falciparum. The inhibitory antibodies targeted both conserved and strain-specific epitopes within the ectodomain of AMA1; however, it appears that the majority of these antibodies reacted with strain-specific epitopes in domain I, the N-terminal disulfide-bonded domain, which is the most polymorphic region of AMA1.
Asunto(s)
Anticuerpos Antiprotozoarios/inmunología , Antígenos de Protozoos , Proteínas de la Membrana/inmunología , Plasmodium falciparum/inmunología , Proteínas Protozoarias/inmunología , Animales , Especificidad de Anticuerpos , Eritrocitos/parasitología , Humanos , Inmunoglobulina G/inmunología , Pliegue de Proteína , Conejos , Proteínas Recombinantes/inmunologíaAsunto(s)
Antígenos de Protozoos , Proteínas de la Membrana/química , Resonancia Magnética Nuclear Biomolecular , Plasmodium falciparum/química , Proteínas Protozoarias/química , Animales , Isótopos de Carbono , Isótopos de Nitrógeno , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , ProtonesRESUMEN
Apical membrane antigen 1 (AMA1) is an asexual blood-stage protein expressed in the invasive merozoite form of Plasmodia species, which are the causative agent of malaria. We have complemented the function of Plasmodium falciparum AMA1 (PfAMA1) with a divergent AMA1 transgene from Plasmodium chabaudi (PcAMA1). It was not possible to disrupt the PfAMA1 gene using 'knock-out' plasmids, although we demonstrate that the PfAMA1 gene can be targeted by homologous recombination. These experiments suggest that PfAMA1 is critical, perhaps essential, for blood-stage growth. Importantly, we showed that PcAMA1 expression in P. falciparum provides trans-species complementation to at least 35% of the function of endogenous PfAMA1 in human red cells. Furthermore, expression of this transgene in P. falciparum leads to more efficient invasion of murine erythrocytes. These results indicate an important role for AMA1 in the invasion of red blood cells (RBCs) across divergent Plasmodium species.
Asunto(s)
Eritrocitos/parasitología , Proteínas de la Membrana/fisiología , Plasmodium chabaudi/fisiología , Plasmodium falciparum/fisiología , Proteínas Protozoarias/fisiología , Animales , Antígenos de Protozoos/fisiología , Antígenos de Superficie/fisiología , HumanosRESUMEN
Although the possibility of a live attenuated malaria vaccine has been considered, current malaria vaccine development activities are dominated by attempts to develop a subunit vaccine. Hence, it is entirely appropriate that a session of the Molecular Approaches to Malaria conference, Lorne, Australia, 2-5 February 2000, was devoted to vaccine development. The oral presentations in this session and the relevant poster presentations are outlined here by Robin Anders and Allan Saul.
Asunto(s)
Vacunas contra la Malaria , Malaria Falciparum/prevención & control , Plasmodium falciparum/inmunología , Proteínas Protozoarias/inmunología , Animales , Antígenos de Protozoos/inmunología , HumanosRESUMEN
Maternally derived antibodies are believed to protect infants against infection, but there is little direct evidence for a protective role of passively acquired antibodies against malaria. A longitudinal study of malaria infection in 143 infants was conducted in a region of southern Ghana where Plasmodium falciparum is endemic. Infants born in the high-transmission season were less likely to become infected in the first 20 weeks of life than children born in the low-transmission season. Plasma, obtained at birth, was tested for immunoglobulin G (IgG) and IgG subclasses to P. falciparum schizonts and recombinant circumsporozoite antigen, MSP-1(19), MSP-2, AMA-1, and Pf155 (also called ring-infected erythrocyte surface antigen). Antibody levels at birth were not associated with resistance to malaria infection. On the contrary, antibodies at birth were positively associated with infection, indicating that high levels of maternally derived antibodies represent a marker for intensity of exposure to malaria infection in infants. However, all five children who experienced high-density infections (>100 parasites/microl of blood) were seronegative for MSP-1(19) at the time of infection.
Asunto(s)
Anticuerpos Antiprotozoarios/sangre , Inmunidad Materno-Adquirida , Malaria Falciparum/prevención & control , Plasmodium falciparum/inmunología , Complicaciones Parasitarias del Embarazo/inmunología , Adolescente , Adulto , Factores de Edad , Animales , Femenino , Ghana/epidemiología , Humanos , Inmunoglobulina G/sangre , Lactante , Recién Nacido , Estudios Longitudinales , Malaria Falciparum/epidemiología , Malaria Falciparum/inmunología , Persona de Mediana Edad , Embarazo , Complicaciones Parasitarias del Embarazo/epidemiología , Complicaciones Parasitarias del Embarazo/parasitología , Prevalencia , Estudios ProspectivosRESUMEN
Apical membrane Ag 1 (AMA1) is a leading malaria vaccine candidate. Homologues of AMA1 can induce protection in mice and monkeys, but the mechanism of immunity is not understood. Mice immunized with a refolded, recombinant, Plasmodium chabaudi AMA1 fragment (AMA1B) can withstand subsequent challenge with P. chabaudi adami. Here we show that CD4+ T cell depletion, but not gammadelta T cell depletion, can cause a significant drop in antiparasite immunity in either immunized normal or immunized B cell KO mice. In normal mice, this loss of immunity is not accompanied by a decline in Ab levels. These observations indicate a role for AMA1-specific Ab-independent T cell-mediated immunity. However, the loss of immunity in normal CD4+ T cell-depleted mice is temporary. Furthermore, immunized B cell KO mice cannot survive infection, demonstrating the absolute importance of B cells, and presumably Ab, in AMA1-induced immunity. CD4+ T cells specific for a cryptic conserved epitope on AMA1 can adoptively transfer protection to athymic (nu/nu) mice, the level of which is enhanced by cotransfer of rabbit anti-AMA1-specific antisera. Recipients of rabbit antisera alone do not survive. Some protected recipients of T cells plus antisera do not develop their own AMA 1-specific Ab response, suggesting that AMA 1-specific CMI alone can protect mice. These data are the first to demonstrate the specificity of any protective CMI response in malaria and have important implications for developing a malaria vaccine.
Asunto(s)
Anticuerpos Antiprotozoarios/fisiología , Antígenos de Protozoos/inmunología , Linfocitos T CD4-Positivos/inmunología , Vacunas contra la Malaria/inmunología , Malaria/inmunología , Malaria/prevención & control , Proteínas de la Membrana/inmunología , Plasmodium chabaudi/inmunología , Proteínas Protozoarias/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antiprotozoarios/biosíntesis , Especificidad de Anticuerpos , Antígenos de Protozoos/administración & dosificación , Antígenos de Protozoos/genética , Linfocitos B/inmunología , Linfocitos T CD4-Positivos/metabolismo , Epítopos de Linfocito T/inmunología , Femenino , Sueros Inmunes/administración & dosificación , Inmunidad Innata , Inyecciones Intraperitoneales , Depleción Linfocítica , Linfopenia/genética , Linfopenia/inmunología , Malaria/parasitología , Vacunas contra la Malaria/administración & dosificación , Proteínas de la Membrana/administración & dosificación , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Desnudos , Datos de Secuencia Molecular , Parasitemia/inmunología , Parasitemia/prevención & control , Plasmodium chabaudi/genética , Proteínas Protozoarias/administración & dosificación , Proteínas Protozoarias/genética , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/inmunologíaRESUMEN
The ring-infected erythrocyte surface antigen (RESA) is a dense-granule protein of Plasmodium falciparum which binds to the cytoskeletal structure of the erythrocyte after parasite invasion. It is currently under trial as a vaccine candidate. In an effort to characterize further the antibody responses to this antigen, we have panned two independent libraries of random peptides expressed on the surface of filamentous phage with a monoclonal antibody (MAb 18/2) against RESA. One library consisted of a potentially constrained 17-mer peptide fused with the gpVIII phage coat protein, and the other displayed an unconstrained 15-mer as a fusion with the minor phage coat protein gpIII. Several rounds of biopanning resulted in enrichment from both libraries clones that interacted specifically with MAb 18/2 in protein-blotting and enzyme-linked immunosorbent assay experiments. Nucleotide sequencing of the random oligonucleotide insert revealed a common predominant motif: (S/T)AVDD. Several other clones had related but degenerate motifs. Thus, a monoclonal antibody against a malarial antigen can select common mimotopes from different random peptide libraries. We envisage many uses for this technology in malaria research.
Asunto(s)
Antígenos de Protozoos/inmunología , Antígenos de Superficie/inmunología , Epítopos de Linfocito B/inmunología , Péptidos/inmunología , Plasmodium falciparum/inmunología , Proteínas Protozoarias/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Antiprotozoarios/inmunología , Bacteriófagos , Ensayo de Inmunoadsorción Enzimática , Humanos , Malaria/inmunología , Malaria/parasitología , Imitación Molecular , Datos de Secuencia Molecular , Biosíntesis de Péptidos , Biblioteca de PéptidosRESUMEN
Two phase I vaccine trials were conducted to test the immunogenicity and safety of a vaccine containing three recombinant malaria antigens from the asexual stage of Plasmodium falciparum. The three antigens are a fragment of MSP1 (190LCS.T3); MSP2 and a portion of RESA and were formulated in Montanide ISA720 adjuvant. These trials investigated the dose response of each antigen for eliciting both antibody and T-cell responses and the immunogenicity of a mixture of the antigens compared with the antigens injected separately. All three antigens elicited both antibody and T-cell responses. Strong T-cell responses were observed with 190LCS.T3 and RESA with stimulation indices exceeding 100 for peripheral blood leucocytes in some individuals. The antibody responses were generally weak. The human antibody responses observed with MSP2 in Montanide ISA720 were not significantly different from those obtained in an earlier trial which used MSP2 with alum as the adjuvant. No antigenic competition was observed: volunteers receiving a mixture of antigens had similar responses to those receiving the three antigens at separate sites. Tenderness and pain at the injection site were common over the first few days following immunization. In some volunteers, especially those receiving the highest doses tested, there was a delayed reaction at the injection site with pain and swelling occurring approximately 10 days after injection.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Antígenos de Protozoos/inmunología , Vacunas contra la Malaria/administración & dosificación , Manitol/análogos & derivados , Ácidos Oléicos/administración & dosificación , Adulto , Animales , Anticuerpos Antiprotozoarios/biosíntesis , Femenino , Cobayas , Humanos , Inmunización Secundaria , Activación de Linfocitos/inmunología , Vacunas contra la Malaria/efectos adversos , Vacunas contra la Malaria/inmunología , Vacunas contra la Malaria/toxicidad , Malaria Falciparum/inmunología , Malaria Falciparum/prevención & control , Masculino , Manitol/administración & dosificación , Ratones , Ratones Endogámicos BALB C , Proteínas Protozoarias/inmunología , Método Simple Ciego , Linfocitos T/inmunologíaRESUMEN
The Plasmodium merozoite surface antigen apical membrane antigen-1 (AMA-1) has previously been shown to provide partial protection to Saimiri and rhesus monkeys immunised with recombinant Plasmodium fragile or parasite-derived Plasmodium knowlesi AMA-1, respectively. In the study reported here we have used the Plasmodium chabaudi/mouse model system to extend our pre-clinical assessment of an AMA-1 vaccine. We describe here the expression of the full-length Plasmodium chabaudi adami AMA-1 and the P. chabaudi adami AMA-1 ectodomain using both baculovirus and Escherichia coli. The ectodomain expressed in E. coli, which contained an N-terminal hexa-his tag, was purified by Ni-chelate chromatography and refolded in vitro in the presence of oxidised and reduced glutathione to generate intramolecular disulphide bonds. In a series of vaccine trials, in both inbred and outbred mice, highly significant protection was obtained by immunising with the refolded AMA-1 ectodomain. Protection was shown to correlate with antibody response and was dependent on intact disulphide bonds. Passive transfer of antibodies raised in rabbits against the refolded AMA-1 ectodomain was also protective. In view of this demonstration that E. coli expression of a soluble P. chabaudi AMA-1 domain can generate a vaccine that is effective in mice, we are pursuing a similar approach to generating a vaccine against P. falciparum for testing in human volunteers.
Asunto(s)
Antígenos de Protozoos , Vacunas contra la Malaria/administración & dosificación , Malaria/prevención & control , Proteínas de la Membrana/administración & dosificación , Plasmodium chabaudi/inmunología , Proteínas Protozoarias/administración & dosificación , Animales , Anticuerpos Antiprotozoarios/inmunología , Malaria/inmunología , Proteínas de la Membrana/biosíntesis , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Proteínas Protozoarias/biosíntesis , Proteínas Recombinantes/administración & dosificaciónRESUMEN
Phage display of single chain variable fragment (scFv) antibodies is a powerful tool for the selection of important and useful antibody specificities. We have constructed such a library from mice protected from malaria challenge by immunization with recombinant Plasmodium chabaudi DS apical membrane antigen (AMA-1). Panning on refolded AMA-1 enriched a population of scFvs which specifically bound the antigen. The single chain antibodies recognize conformational epitopes on AMA-1 from the P. chabaudi DS strain but not on AMA-1 of the 556KA strain of P. chabaudi. A subset of the antibody fragments recognized AMA-1 from the human malaria parasite Plasmodium falciparum. Nucleotide sequencing revealed that at least four unique scFv genes were selected by the panning procedure. These scFv antibodies are valuable reagents for probing the structure and function of AMA-1 and will be used to test the feasibility of using recombinant antibodies in a passive immunization therapy against malaria.
Asunto(s)
Anticuerpos Antiprotozoarios/inmunología , Antígenos de Protozoos/inmunología , Fragmentos de Inmunoglobulinas/aislamiento & purificación , Región Variable de Inmunoglobulina/aislamiento & purificación , Vacunas contra la Malaria/inmunología , Proteínas de la Membrana/inmunología , Proteínas Protozoarias/inmunología , Animales , Antígenos de Superficie/inmunología , Bacteriófago M13 , Ensayo de Inmunoadsorción Enzimática , Mapeo Epitopo , Escherichia coli , Humanos , Fragmentos de Inmunoglobulinas/inmunología , Región Variable de Inmunoglobulina/inmunología , Ratones , Biblioteca de Péptidos , Plasmodium chabaudi/inmunología , Plasmodium falciparum/inmunologíaRESUMEN
Various formulations of the Plasmodium falciparum merozoite surface antigen, MSA-2, were made and tested in animals in order to select one for use in human vaccine trials. Recombinant constructs representing both major allelic forms of MSA-2 were formulated with a range of adjuvants and used to immunize rabbits, mice and sheep. After immunization, antibody responses obtained with the most potent adjuvants were at least tenfold greater than responses obtained with the least potent adjuvant Alhydrogel, which was used as the reference standard, although its lower potency indicated against its further use in clinical trials. Based on broadly similar results obtained with the three animal species, several adjuvants, including the water-in-oil adjuvant Montanide ISA 720, the oil-in-water adjuvant SAF-1, and liposomes containing lipid A formulated with Alhydrogel were demonstrated to be potent and potentially suitable for the clinical evaluation of MSA-2 as a candidate malaria vaccine antigen. Of these, ISA 720 was selected for further trial.
Asunto(s)
Adyuvantes Inmunológicos/uso terapéutico , Antígenos de Protozoos , Vacunas contra la Malaria/inmunología , Proteínas Protozoarias/inmunología , Animales , Anticuerpos Antiprotozoarios/biosíntesis , Anticuerpos Antiprotozoarios/inmunología , Antígenos de Superficie/inmunología , Evaluación Preclínica de Medicamentos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Plasmodium falciparum/inmunología , Conejos , Ovinos , Especificidad de la EspecieRESUMEN
The prevalence and concentration of IgG antibodies to defined Plasmodium falciparum antigens were assessed in serum samples of 97 children with cerebral malaria and 146 children with uncomplicated malaria. The antigens used included the schizont extract, ring-infected erythrocyte surface antigen, the C-terminal region of merozoite surface antigen-1 (MSA-1) (BVp42), and three recombinant proteins of MSA-2 (FC27, 3D7, and d3D7). Parasite isolates from 24 children with cerebral malaria and 22 children with uncomplicated malaria were genotyped for MSA-1 and MSA-2. The distribution of parasite genotypes belonging to the different allelic families was similar in both the cerebral and uncomplicated malaria groups. There were higher antibody levels to antigens derived from the infecting parasite genotype than to heterologous genotypes, but this difference was only statistically significant for antibody against the d3D7 antigen among children infected with the 3D7 parasite genotype (mean log = 4.72 versus 3.45 antibody units [AU]; P = 0.029). Those who died were more likely to be infected with the FC27 genotype and had lower antibody levels to MSA-2 of the 3D7 type than had cerebral malaria patients who survived (mean log = 2.94 versus 3.79 AU; P = 0.049). Antibodies against parasites of the 3D7 genotype are associated with a better prognosis among children with cerebral malaria partly because these children are more likely to be infected with parasites of this genotype rather than the FC27 genotype, which appears to be more virulent.
Asunto(s)
Anticuerpos Antiprotozoarios/biosíntesis , Antígenos de Protozoos/inmunología , Malaria Cerebral/inmunología , Malaria Falciparum/inmunología , Plasmodium falciparum/inmunología , Enfermedad Aguda , Adolescente , Animales , Antígenos de Superficie/genética , Antígenos de Superficie/inmunología , Niño , Preescolar , Femenino , Genotipo , Hemoglobinas/análisis , Humanos , Inmunoglobulina G/biosíntesis , Lactante , Recuento de Leucocitos , Malaria Cerebral/parasitología , Malaria Falciparum/parasitología , Masculino , Proteína 1 de Superficie de Merozoito , Plasmodium falciparum/genética , Precursores de Proteínas/genética , Precursores de Proteínas/inmunología , Proteínas Protozoarias/genética , Proteínas Protozoarias/inmunologíaRESUMEN
Isolates of Plasmodium falciparum obtained from 12 children attending different health facilities in the Madang Province, Papua New Guinea were typed for allelic variants of merozoite surface protein-1 and merozoite surface protein-2. Blood was obtained just before treatment with either amodiaquine or chloroquine and at intervals following treatment. All patients examined were found to be infected with genetically different parasites. Nine of the children were found to have single infections while three had mixed infections. In all patients, parasites reappearing in the blood following treatment had the same genotype as parasites in the primary infection. These results indicate that parasites reappearing in the blood following treatment were the result of true recrudescence and not new infections.
Asunto(s)
Antígenos de Protozoos , Malaria Falciparum/parasitología , Plasmodium falciparum/efectos de los fármacos , Alelos , Animales , Niño , Preescolar , Resistencia a Medicamentos/genética , Femenino , Frecuencia de los Genes , Variación Genética , Genotipo , Humanos , Masculino , Proteína 1 de Superficie de Merozoito , Plasmodium falciparum/clasificación , Plasmodium falciparum/genética , Precursores de Proteínas/genética , Proteínas Protozoarias/genética , RecurrenciaRESUMEN
An 8kb gene coding for a putative serine/threonine protein kinase from Plasmodium falciparum has been cloned and sequenced. It is arranged in two exons: exon I is 2 kb and exon II is 5.6 kb. The gene codes for a large protein of 2510 amino acids. Antibodies raised against a fusion protein were used to localize the putative kinase. By immunofluorescence microscopy, it was found in the cytoplasm of infected red cells. By immunoelectron microscopy it was associated with membranous structures in the red cell and with the red cell membrane, particularly at parasite-induced knobs. This is the first putative protein kinase of P. falciparum to be exported from the parasite into its host cell.
Asunto(s)
Genes Protozoarios , Plasmodium falciparum/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Protozoarias/genética , Secuencia de Aminoácidos , Animales , Antígenos de Superficie/aislamiento & purificación , Compartimento Celular , Clonación Molecular , Membrana Eritrocítica/ultraestructura , Escherichia coli/genética , Técnica del Anticuerpo Fluorescente , Microscopía Inmunoelectrónica , Datos de Secuencia Molecular , Plasmodium falciparum/enzimología , Plasmodium falciparum/ultraestructura , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Protozoarias/aislamiento & purificación , Proteínas Protozoarias/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Análisis de Secuencia de ADNRESUMEN
A prospective community study in a highly malaria endemic area of Papua New Guinea found that infection with multiple Plasmodium falciparum genotypes was an indicator of lowered risk of subsequent clinical attack. The results suggest that concurrent or very recent infections provide protection from superinfecting parasites. The finding of an association between reduced risk of clinical malaria and infection with parasites of merozoite surface protein 1 (MSP-1) type RO33 or MSP-2 type 3D7 further suggests that the concomitant immunity is, at least in part, a consequence of a response to these major merozoite surface proteins.
Asunto(s)
Enfermedades Endémicas , Malaria Falciparum/epidemiología , Plasmodium falciparum/clasificación , Adolescente , Distribución por Edad , Animales , Niño , Preescolar , Estudios de Cohortes , Estudios Transversales , Genotipo , Humanos , Lactante , Malaria Falciparum/parasitología , Morbilidad , Papúa Nueva Guinea/epidemiología , Plasmodium falciparum/genética , Reacción en Cadena de la Polimerasa , Prevalencia , Estudios ProspectivosRESUMEN
Merozoite surface protein-3 (MSP-3) is a secreted polymorphic antigen associated with erythrocytic schizonts and merozoites of Plasmodium falciparum asexual blood-stages. A prominent structural feature of MSP-3 is a domain composed of three blocks of tandemly-repeated heptads with the consensus sequence AXXAXXX. The three blocks of four alanine heptad-repeats are separated by short stretches of non-repetitive sequence unrelated to the heptad-repeat. C-terminal to the heptad-repeats, MSP-3 contains a glutamic acid-rich domain followed by another heptad-repeat similar to a leucine-zipper motif. An analysis of the msp-3 gene from four P. falciparum isolates shows that polymorphism in MSP-3 is predominantly due to sequence diversity in the N-terminal half of the predicted polypeptide within and flanking the heptad-repeats. Mutations in the region of the gene that encodes the alanine heptad-repeats appear to be of two types. Unique mutations in non-repetitive sequence have generated amino acid substitutions and deletions that result in unique sequences among MSP-3 variants. In contrast, mutations in the heptad-coding sequence are largely dimorphic and are clustered in one or two heptads in each of the three blocks of heptads. Despite the diversity within and flanking the heptad domain the AXXAXXX motif is highly conserved as are other features of the sequence that predict the formation of alpha-helical secondary structure. Recombinant proteins and a synthetic peptide were used to raise antisera to conserved and variable regions of MSP-3. Differential reactivity of these reagents with the parasite antigen identified the alanine heptad-repeat domain as a site of antigenic diversity among MSP-3 polypeptides.
Asunto(s)
Variación Antigénica , Antígenos de Protozoos/química , Plasmodium falciparum/inmunología , Proteínas Protozoarias/química , Proteínas Protozoarias/inmunología , Secuencia de Aminoácidos , Animales , Antígenos de Protozoos/genética , Antígenos de Protozoos/inmunología , Secuencia Conservada , Genes Protozoarios , Datos de Secuencia Molecular , Mutación , Plasmodium falciparum/genética , Polimorfismo Genético , Estructura Secundaria de Proteína , Proteínas Protozoarias/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/inmunologíaRESUMEN
We have investigated the proliferative and Th cell responses to the Plasmodium chabaudi adami DS homologue of the Plasmodium falciparum apical membrane Ag 1 (AMA-1), a leading malaria vaccine candidate. Immunodominant T cell epitopes were defined following immunization of BALB/c mice with Escherichia coli-expressed, refolded P. c. adami DS AMA-1 recombinant protein and testing cells from the draining lymph nodes for responses against a series of overlapping peptides spanning P. c. adami AMA-1. A limited number of major T cell sites were identified in both conserved and variable regions of the protein. Several cryptic epitopes that evoked T cell responses following immunization with peptides, but not after protein immunization, were also identified. Adoptive transfer of a T cell line specific for a conserved cryptic epitope (corresponding to residues 31-50) provided help for an anti-AMA-1 protein-specific Ab response following in vivo challenge with P. c. adami parasitized RBC, such that AMA-1-specific Abs appeared more rapidly in recipient mice than in controls. Furthermore, T cells specific for cryptic epitopes afforded partial protection against P. c. adami infection in nude mice. The identification of conserved cryptic Th cell epitopes has important implications for malaria vaccine design.